Methods and compositions for preparing tagatose from fructose

ABSTRACT

Disclosed herein are compositions and methods for preparing tagatose from fructose, more particularly, compositions comprising thermophilic fructose C4-epimerases derived from thermophilic microorganisms and methods for preparing tagatose from fructose using the compositions.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority to, and the benefits of U.S.Provisional Patent Application No. 62/693,681, filed Jul. 3, 2018, and62/693,660, filed Jul. 3, 2018, each of which is herein incorporated byreference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is ARZE_028_01US_SeqList_ST25.txt. The text file isabout 1.56 megabytes, was created on Jul. 3, 2019 and is being submittedelectronically via EFS-Web.

FIELD OF THE INVENTION

The invention described herein generally relates to the fields ofmicrobiology, biochemistry, and organic chemistry.

BACKGROUND

As problems with obesity and diabetes continue to rise globally, it isof growing importance to have access to alternative low-caloriesweeteners that provide health benefits over traditional sweeteners.D-tagatose is a naturally occurring sweetener that is GRAS (generallyrecognized as safe) for use in food and beverages. It is 92% as sweet assucrose and has less than half the caloric value. Importantly,D-tagatose has a taste profile very similar to sucrose and does not havea lingering bitterness characteristic of related alternative sugars suchas psicose (allulose). D-tagatose has been suggested to have severalpositive health benefits including non-tooth decaying,anti-hyperglycemic, and a positive prebiotic effect.

The ketohexose monosaccharide D-tagatose is a C4-epimer of D-fructose.Therefore, D-tagatose is a reducing sugar that undergoes browningreactions and can be used as a “drop-in” replacement for typical bulksweeteners. There exist established methods to produce D-tagatose fromgalactose (commonly produced from lactose) using both chemical andbiocatalytic means. However, the high cost of generating the galactosefeedstock makes the galactose route less attractive than a process thatcan utilize lower-cost feedstocks such as fructose, glucose, or sucrose.

SUMMARY OF THE INVENTION

The present disclosure provides compositions and methods for producingtagatose by epimerizing fructose at carbon number 4. The presentdisclosure allows the use of fructose as the source material fortagatose production, which is more economical than using galactose asthe source material.

In one aspect, the disclosure provides a polypeptide comprising an aminoacid sequence having at least 60% sequence identity to any one of SEQ IDNOs:1-444, wherein the polypeptide has D-fructose C4-epimerase activity.In further embodiments, the polypeptide comprises an amino acid sequencethat is at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.5%, or at least 99.9% identical to any one of SEQ ID NOs: 1-444,wherein the polypeptide has D-fructose C4-epimerase activity.

In other embodiments, the polypeptide comprises one or moremodifications at the amino acid residue corresponding to position L3,S4, K5, D6, Y7, L8, R9, K10, K11, V13, Y14, S15, I16, C17, S18, S19,S21, Y22, E25, S27, E29, F30, K32, E33, K34, G35, D36, Y37, E41, T43,P44, H45, Q46, Q49, F50, Y53, S54, M56, E59, K62, N63, F64, M66, K67,K70, E71, K72, E75, E76, D77, K78, I80, D84, H85, L89, Q92, D93, E94,S96, P97, T98, N101, K102, K104, D105, R108, F110, S113, Y115, K116,K117, H119, D121, C122, S123, M124, P125, L126, S127, D128, D129, P130,K131, V132, P134, Y135, E136, K137, E140, R141, T142, R143, E144, F146,E147, I148, E150, E151, T152, A153, R154, K155, Y156, N157, F158, Q159,P160, V161, T166, D167, V168, P169, I170, A171, G172, G173, G174, E175,E176, E177, G178, V179, V182, D184, S187, A188, I189, S190, S191, L192,K193, K194, Y195, N197, D198, V199, P200, N201, I202, W203, D204, R205,G208, V210, I211, M212, L213, I215, G216, F217, S218, Y219, D220, K221,V222, E224, D228, K229, V230, R231, G232, I233, L234, E235, V237, K238,R239, E240, D241, L242, V244, E245, G246, H247, S248, T249, D250, A253,Y255, A256, R258, N259, E262, R266, K269, V270, G271, P272, A273, R280,G281, V282, L284, S286, N287, D290, E291, I292, E295, R296, E297, S299,N300, K302, R303, E307, D313, Y315, K318, Y319, K321, D322, S323, K324,R325, L326, E327, L328, I330, W331, N333, L334, L335, D336, R337, R339,Y340, E343, Y344, E345, K348, M349, V350, N352, K353, E356, N357, F358,E360, G361, V362, D363, I364, K365, F366, Y368, Q369, Y370, Y372, D373,S374, Y375, F376, D377, R379, E380, K382, M383, K384, N385, D386, R388,E389, K392, K393, K396, R397, E400, S403, N407, or L408 of SEQ ID NO:6;or combinations thereof.

In some embodiments, the polypeptide comprises one or more ofmodifications: L3E, L3T, S4M, S4D, S4R, S4L, S4Q, S4N, S4E, S4P, K5F,K5M, K5L, K5R, K5W, K5H, K5Q, K5V, K5A, D6R, D6L, D6H, D6Q, D6N, D6A,D6E, Y7W, Y7H, Y7N, L8N, L8G, R9L, R9T, R9H, R9Q, R9E, R9I, K10M, K10D,K10R, K10L, K10V, K10N, K10A, K10E, K10P, K11F, K11L, K11R, K11Y, K11W,K11V, K11Q, K11H, K11E, K11I, V13I, Y14F, S15A, I16V, C17S, C17L, C17R,C17T, C17G, C17V, C17A, C17I, S18N, S18A, S19N, S21K, S21R, S21Q, S21E,S21P, Y22W, E25R, E25I, S27C, S27A, E29Q, E29R, E29I, E29L, F30H, F30T,F30A, K32S, K32M, K32L, K32R, K32Q, K32A, K32I, E33R, E33T, E33H, E33Q,E33N, E33D, K34F, K34S, K34M, K34R, K34L, K34Y, K34T, K34H, K34Q, K34V,K34E, K34D, K34I, G35D, G35N, G35H, G35S, D36F, D36S, D36R, D36L, D36Y,D36T, D36W, D36H, D36Q, D36N, D36E, Y37D, Y37P, Y37R, Y37E, E41Q, E41A,H45F, H45K, H45Q, H45A, Q46S, Q46R, Q46T, Q46W, Q46A, Q49F, Q49H, Q49I,Q49W, F50Q, F50H, F50R, F50S, Y53F, Y53V, Y53S, Y53A, S54T, M56W, E59R,E59S, E59T, K62V, K62R, K62Y, K62A, N63E, N63D, N63R, N63Q, F64E, F64L,M66F, M66E, M66Y, K67S, K67R, K67T, K67Q, K67E, K67D, K70D, K70Q, K70R,K70L, E71Q, E71R, E71S, K72L, K72R, K72T, K72V, K72Q, K72H, K72E, K72I,E75D, E75P, E75R, E75S, E76P, E76L, E76R, D77N, D77R, D77S, K78M, K78R,K78L, K78Q, K78D, I80Q, I80R, I80L, D84P, D84N, D84G, D84A, H85A, L89S,L89G, Q92M, Q92D, Q92G, Q92A, Q92P, D93H, E94Q, E94R, E94Y, E94L, S96E,S96R, S96A, P97E, P97D, T98R, T98W, T98V, T98Q, T98E, T98I, N101S,N101D, N101R, N101T, N101H, N101Q, N101A, N101E, N101K, K102L, K102R,K102Y, K102W, K102Q, K102E, K104F, K104R, K104V, K104Q, K104N, K104E,K104I, D105E, D105Q, D105I, R108I, F110Y, S113A, Y115F, K116S, K116R,K116L, K116Y, K116T, K116W, K116H, K116Q, K116E, K116D, K116I, K117M,K117L, K117R, K117W, K117V, H119S, H119G, H119N, H119A, H119P, D121N,D121A, C122R, C122G, C122T, C122V, C122A, C122E, C122K, S123M, S123R,S123T, S123A, M124K, M124R, P125A, L126C, S127A, D128G, D129R, D129L,D129W, D129Q, D129E, P130S, K131S, K131R, K131L, K131Y, K131Q, K131A,K131E, K131D, V132S, V132R, V132Y, V132T, V132E, P134D, P134H, P134A,Y135K, Y135M, Y135D, Y135L, Y135R, Y135T, Y135W, Y135Q, Y135N, Y135P,E136S, E136R, E136P, E136W, E136T, E136Q, E136D, K137L, K137R, K137T,K137W, K137V, K137Q, K137E, K137I, E140D, E140R, E140I, E140L, R141L,R141Y, R141W, R141H, R141Q, R141D, T142E, R143V, R143A, E144Q, E144D,E144R, E144I, F146C, E147Q, E147K, E147R, E147A, I148E, I148V, I148D,E150R, E150A, E151R, T152R, T152Y, T152V, T152H, T152Q, T152A, A153Q,A153R, A153G, A153S, R154S, R154T, R154Q, R154E, R154P, K155R, K155Y,K155P, K155T, K155H, K155N, K155Q, K155G, K155W, K155E, K155D, Y156F,Y156M, Y156R, Y156T, Y156V, Y156Q, Y156E, Y156D, N157E, N157D, N157R,N157P, F158R, F158L, F158T, F158V, F158N, F158E, Q159R, Q159L, Q159G,Q159T, Q159W, Q159E, P160F, P160S, P160M, P160L, P160R, P160T, P160A,P160E, P160I, V161E, V161I, V161Y, V161W, T166M, T166L, T166I, D167T,D167V, D167Q, D167N, D167A, V168M, V168R, I170M, I170K, I170T, A171P,G173S, E175K, E175V, E175R, E176F, E176Y, G178P, V179R, V179I, V182P,V182K, V182A, D184E, S187R, S187T, S187N, S187Q, S187A, S187E, S187K,A188M, A188F, A188T, I189L, S190E, S190D, S190Y, S191L, S191R, S191Y,S191T, S191W, S191V, S191H, S191Q, S191A, S191E, S191I, L192F, L192H,K193F, K193R, K193L, K193Y, K193Q, K193A, K193E, K193I, K194S, K194R,K194L, K194T, K194Q, K194A, K194E, K194D, Y195H, Y195Q, Y195A, N197S,N197D, N197R, N197L, N197P, N197Q, N197A, N197E, N197K, D198K, V199H,V199L, V199I, V199C, P200M, P200L, P200R, P200G, P200E, P200D, N201Q,N201K, N201R, N201E, I202M, I202A, W203F, W203R, W203L, W203Y, W203H,W203A, D204N, D204R, D204S, R205Q, R205L, G208A, V210P, V210T, I211V,M212K, M212L, M212R, L213M, I215V, G216N, G216R, G216T, S218T, S218G,Y219D, Y219H, Y219R, Y219G, D220Q, D220R, D220E, D220T, K221R, K221T,K221V, K221Q, K221E, K221I, V222I, E224M, E224Q, E224Y, D228E, D228S,K229S, K229R, K229T, K229N, K229Q, K229A, K229E, V230A, R231E, R231L,R231F, R231A, G232D, G232R, G232P, G232E, G232K, I233L, L234M, L234A,E235W, E235R, V237L, K238S, K238R, K238W, K238Q, K238E, R239S, E240M,E240Y, E240T, E240V, E240N, E240H, E240D, D241R, D241G, D241Q, D241N,D241P, L242W, V244L, E245Q, E245A, G246C, G246V, G246A, H247A, T249S,D250N, D250A, A253S, A253P, A253T, A253N, A253D, Y255E, Y255D, Y255Q,Y255K, A256L, R258E, R258L, N259R, N259W, N259A, N259E, N259D, E262Q,E262R, E262L, R266V, R266A, K269L, K269R, K269V, K269N, K269I, G271D,G271A, A273W, R280E, G281A, V282I, L284K, S286D, S286V, S286A, N287F,N287R, N287L, N287H, N287Q, N287E, N287D, D290F, D290R, D290Q, D290N,D290A, D290E, D290K, E291D, E291R, I292Q, I292L, I292E, E295P, R296E,R296D, R296Y, E297R, E297L, E297G, E297H, E297Q, E297K, S299Q, S299A,N300E, N300D, N300Q, N300R, K302M, K302L, K302R, K302Q, K302E, K302P,K302I, R303E, R303D, R303Q, E307Q, E307R, D313P, Y315W, K318E, K318D,K318R, K318N, K321S, K321R, K321T, K321H, K321E, K321D, D322T, S323D,S323G, K324S, K324R, K324P, K324W, K324H, K324E, K324D, R325S, R325G,R325W, R325N, R325Q, R325A, R325E, R325D, L326H, E327R, E327I, E327Y,L328F, L328R, L328Y, L328W, L328E, L328D, I330L, W331E, W331H, W331L,W331F, N333A, N333S, L334Y, R339E, R339T, R339A, R339G, Y340F, Y340R,Y340H, Y340A, Y340P, E343M, E343L, E343Y, E343Q, E343N, E343D, Y344H,E345D, E345P, K348R, K348L, K348Y, K348W, K348V, K348Q, K348A, K348E,M349R, M349Y, M349C, M349T, M349W, M349N, M349Q, M349A, M349E, M349K,M349I, V350A, N352M, N352R, N352L, N352Q, N352E, K353R, K353L, K353W,K353H, K353Q, K353A, K353E, E356W, E356R, N357H, F358L, E360D, G361D,G361P, V362I, D363P, D363H, I364M, I364L, I364W, I364E, I364P, K365S,K365R, K365T, K365H, K365N, K365E, K365D, F366H, F366L, F366R, F366Y,Y368R, Y368L, Q369E, Q369D, Q369R, Q369L, Y370H, Y372P, D373R, D373L,D373N, D373A, D373E, S374C, S374Q, S374T, S374A, Y375W, Y375A, F376R,F376H, F376Q, F376E, F376K, D377S, D377R, D377H, D377N, D377A, R379C,E380D, E380N, E380R, E380L, K382R, K382L, K382N, K382Q, K382E, K382D,M383L, K384S, K384L, K384R, K384T, K384Q, K384E, K384D, N385P, N385T,D386H, R388E, E389D, E389R, K392F, K392M, K392R, K392L, K392Y, K392H,K392E, K393F, K393L, K393R, K393Y, K393T, K393W, K393H, K393Q, K393A,K393E, K396F, K396M, K396L, K396R, K396Q, K396A, K396E, K396I, R397S,R397L, R397H, R397Q, R397E, R397K, R397I, E400Q, E400R, E400Y, E400L,S403F, S403R, S403L, S403Y, S403T, S403W, S403H, S403A, S403E, N407F,N407R, N407L, N407Y, N407W, N407H, N407Q, N407E, N407D, N407I, L408R,L408T, L408G, L408W, L408N, or L408D; or combinations thereof.

In other embodiments, the polypeptide comprises one or more ofmodifications at the amino acid residue corresponding to position C17,S18, E41, T43, P44, H45, Q46, Y53, D84, H85, L89, Q92, H119, D121, C122,S123, M124, T166, D167, V168, P169, I170, A171, G172, G173, G174, E175,E176, E177, G178, V210, M212, F217, E245, H247, S248, T249, D250, K269,V270, G271, P272, Y319, L335, D336, R337, R339, or Y340 of SEQ ID NO: 6;or combinations thereof.

In some embodiments, the polypeptide comprises one or more ofmodifications: C17A, C17T, S18A, S18N, E41A, E41Q, H45F, H45A, H45K,H45Q, Q46R, Q46S, Q46A, Q46W, Q46T, Y53F, Y53A, Y53S, Y53V, D84G, D84A,D84N, D84P, H85A, L89G, L89S, Q92M, Q92D, Q92P, Q92G, Q92A, H119N,H119S, H119P, H119G, H119A, D121A, D121N, C122G, C122K, C122E, S123A,S123M, S123R, S123T, M124K, M124R, T166L, T166M, T166I, D167N, D167V,D167A, D167Q, D167T, V168M, V168R, I170M, I170K, I170T, A171P, G173S,E175K, E175V, E175R, E176F, E176Y, G178P, V210P, V210T, M212L, M212K,M212R, E245A, E245Q, H247A, T249S, D250A, D250N, G271D, G271A, R339G,R339A, R339E, R339T, Y340F, Y340H, Y340R, Y340P, or Y340A; orcombinations thereof.

In other embodiments, the polypeptide comprises one or more ofmodifications at the amino acid residue corresponding to position V13,Y14, S15, I16, S19, S21, Y22, S27, F30, K34, G35, D36, Y37, S54, K62,N63, M66, K67, K72, E75, E76, D77, K78, D93, S96, P97, T98, N101, D105,F110, S113, Y115, L126, S127, D128, D129, K131, Y135, E136, E140, T142,R143, E144, F146, E147, I148, E151, T152, R154, P160, V179, V182, D184,S187, A188, I189, S190, S191, L192, Y195, N197, D198, V199, P200, N201,I202, D204, G208, I211, L213, I215, S218, Y219, V230, G232, I233, L234,V237, E240, D241, L242, V244, G246, A253, Y255, A256, N259, E262, R266,A273, R280, G281, V282, L284, S286, N287, D290, E291, I292, E295, R296,E297, K302, R303, K321, K324, R325, E327, L328, I330, W331, N333, L334,E343, Y344, E345, M349, V350, N352, K353, F358, E360, V362, D363, I364,K365, F366, Y372, D373, S374, F376, D377, R379, M383, N385, K393, R397,or S403 of SEQ ID NO:6; or combinations thereof.

In some embodiments, the polypeptide comprises one or more ofmodifications: V13I, Y14F, S15A, I16V, S19N, S21P, S21E, S21K, Y22W,S27A, S27C, F30H, K34D, K34M, K34R, K34Y, G35D, G35H, D36T, D36S, Y37P,S54T, K62R, N63R, M66F, M66Y, K67Q, K72V, K72L, E75P, E76P, D77N, K78R,D93H, S96E, S96A, P97D, P97E, T98E, T98Q, N101E, N101A, N101K, D105E,F110Y, S113A, Y115F, L126C, S127A, D128G, D129E, K131A, Y135P, Y135Q,Y135K, Y135N, Y135T, Y135M, Y135D, Y135L, E136D, E140I, T142E, R143V,R143A, E144R, F146C, E147R, E147K, I148V, E151R, T152A, R154Q, P160E,P160A, V179I, V182P, V182A, V182K, D184E, S187K, S187N, S187T, S187E,S187R, A188F, A188M, A188T, I189L, S190D, S190E, S191I, S191T, S191V,S191Y, S191A, S191L, L192F, L192H, Y195A, N197Q, N197K, N197E, N197D,N197A, D198K, V199I, V199H, V199L, V199C, P200G, N201R, N201Q, N201K,I202M, I202A, D204N, G208A, I211V, L213M, I215V, S218G, Y219D, Y219H,V230A, G232D, G232E, G232K, G232P, I233L, L234M, L234A, V237L, E240T,D241P, D241G, L242W, V244L, G246V, G246A, G246C, A253P, A253N, A253T,A253D, A253S, Y255D, Y255E, Y255Q, Y255K, A256L, N259R, N259E, N259W,N259D, N259A, E262Q, R266V, R266A, A273W, R280E, G281A, V282I, L284K,S286V, S286D, S286A, N287D, D290N, D290A, D290Q, D290K, E291D, I292L,E295P, R296D, R296E, R296Y, E297Q, E297K, E297G, E297H, E297R, K302L,R303E, K321T, K321H, K321R, K324D, K324E, R325D, R325N, R325E, R325Q,E327R, L328F, L328W, I330L, W331H, N333A, N333S, L334Y, E343N, Y344H,E345P, M349K, M349C, M349T, M349E, M349R, M349A, V350A, N352E, K353R,F358L, E360D, V362I, D363P, I364W, K365T, K365R, F366L, Y372P, D373A,S374T, S374Q, S374A, S374C, F376Q, F376K, F376E, F376H, F376R, D377A,R379C, M383L, N385P, K393H, R397K, S403H, S403Y, or S403A; orcombinations thereof.

In other embodiments, the polypeptide comprises one or more ofmodifications at the amino acid residue corresponding to position K5,K10, K11, C17, K32, K34, K62, K67, K70, K72, K78, K102, K104, K116,K117, C122, K131, K137, K155, K193, K194, K221, K229, K238, K269, K302,K318, K321, K324, K348, K353, K365, K382, K384, K392, K393, or K396 ofSEQ ID NO:6; or combinations thereof.

In some embodiments, the polypeptide comprises one or more ofmodifications: K5H, K5M, K5L, K5A, K5R, K5W, K5F, K10M, K10L, K10R,K10A, K10V, K10N, K11Q, K11Y, K11E, K11L, K11R, K11V, C17T, C17I, C17L,C17A, C17R, C17V, C17G, C17S, K32Q, K32I, K32M, K32L, K32R, K32S, K34T,K34Q, K34Y, K34E, K34H, K34M, K34L, K34R, K34F, K62R, K62Y, K62V, K67R,K67T, K67Q, K67S, K70R, K70Q, K72T, K72I, K72L, K72R, K72V, K78R, K78Q,K78M, K78L, K102R, K102Y, K102L, K104Q, K104I, K104R, K104V, K104N,K104F, K116T, K116Q, K116Y, K116H, K116I, K116L, K116R, K116W, K116S,K117M, K117L, K117R, K117V, K117W, C122R, C122T, C122A, C122V, K131Q,K131Y, K131E, K131L, K131R, K137T, K137Q, K137E, K137I, K137L, K137R,K155Y, K155E, K155H, K155R, K155D, K155N, K193Q, K193E, K193I, K193R,K193A, K194T, K194E, K194R, K194A, K194S, K221Q, K221T, K221I, K221R,K221V, K229T, K229Q, K229R, K229A, K229N, K229S, K238R, K238W, K238S,K269I, K269L, K269R, K269V, K269N, K302Q, K302I, K302M, K302L, K302R,K318R, K318D, K318E, K321R, K321D, K324R, K324D, K324E, K324S, K348V,K348R, K348L, K353W, K353R, K353Q, K353L, K365R, K365H, K365S, K382R,K382N, K382L, K384T, K384Q, K384E, K384L, K384D, K384R, K384S, K392R,K392M, K392L, K393Y, K393H, K393L, K393R, K393F, K396I, K396M, K396L,K396R, or K396F; or combinations thereof.

In other embodiments, the polypeptide comprises one or more ofmodifications at the amino acid residue corresponding to position L3,S4, K5, D6, Y7, L8, R9, K10, K11, S21, Y22, E25, E29, F30, K32, E33,K34, G35, D36, Y37, Q49, F50, M56, E59, K62, N63, F64, M66, K67, K70,E71, K72, E75, E76, D77, K78, I80, E94, S96, T98, N101, K102, K104,D105, R108, K116, P125, D129, P130, K131, V132, P134, Y135, E136, K137,E140, R141, E144, E147, I148, E150, E151, T152, A153, R154, K155, Y156,N157, F158, Q159, P160, V161, V179, S187, S190, S191, K193, K194, Y195,N197, P200, N201, W203, D204, R205, G216, S218, Y219, D220, K221, V222,E224, D228, K229, R231, G232, E235, K238, R239, E240, D241, R258, E262,N287, D290, E291, I292, E297, S299, N300, K302, R303, E307, D313, Y315,K318, K321, D322, S323, K324, R325, L326, E327, L328, W331, E343, E345,K348, M349, N352, K353, E356, N357, G361, D363, I364, K365, F366, Y368,Q369, Y370, D373, Y375, F376, D377, E380, K382, K384, N385, D386, R388,E389, K392, K393, K396, R397, E400, S403, N407, or L408 of SEQ ID NO:6;or combinations thereof.

In some embodiments, the polypeptide comprises one or more ofmodifications: L3E, L3T, S4L, S4D, S4R, S4N, S4P, S4E, S4Q, S4M, K5L,K5V, K5A, K5H, K5R, K5F, K5Q, D6L, D6A, D6H, D6R, D6N, D6E, D6Q, Y7W,Y7N, Y7H, L8N, L8G, R9L, R9I, R9H, R9E, R9Q, R9T, K10D, K10A, K10R,K10N, K10P, K10E, K11I, K11V, K11H, K11Y, K11R, K11F, K11W, K11E, K11Q,S21R, S21E, S21Q, S21P, Y22W, E25R, E25I, E29L, E29R, E29Q, E29I, F30A,F30H, F30T, K32L, K32I, K32A, K32R, K32Q, K32M, E33D, E33H, E33R, E33N,E33Q, E33T, K34L, K34D, K34I, K34S, K34V, K34H, K34Y, K34R, K34F, K34Q,K34T, K34M, G35D, G35N, G35H, G35S, D36L, D36S, D36H, D36Y, D36R, D36E,D36N, D36F, D36W, D36Q, D36T, Y37E, Y37R, Y37D, Y37P, Q49W, Q491, Q49H,Q49F, F50R, F50Q, F50H, F50S, M56W, E59R, E59S, E59T, K62R, K62A, N63R,N63E, N63D, N63Q, F64L, F64E, M66E, M66F, M66Y, K67D, K67S, K67R, K67E,K67Q, K67T, K70L, K70R, K70D, K70Q, E71R, E71Q, E71S, K72L, K72I, K72H,K72E, K72Q, K72T, E75R, E75D, E75S, E76L, E76R, E76P, D77R, D77N, D77S,K78L, K78R, K78D, K78Q, K78M, I80L, I80R, I80Q, E94L, E94R, E94Q, E94Y,S96R, S96E, S96A, T98I, T98V, T98R, T98W, T98E, T98Q, N101D, N101S,N101A, N101H, N101R, N101E, N101Q, N101T, K102Y, K102R, K102W, K102E,K102Q, K104R, K104E, K104Q, D105E, D105Q, D105I, R108I, K116L, K116I,K116H, K116Y, K116R, K116E, K116W, K116D, K116Q, K116T, P125A, D129L,D129R, D129W, D129E, D129Q, P130S, K131D, K131E, K131S, K131Y, V132S,V132Y, V132R, V132E, V132T, P134A, P134D, P134H, Y135L, Y135D, Y135R,Y135W, Y135Q, E136S, E136R, E136P, E136W, E136D, E136Q, E136T, K137L,K137I, K137V, K137R, K137W, K137E, K137Q, E140L, E140R, E140D, E140I,R141L, R141D, R141H, R141Y, R141W, R141Q, E144R, E144Q, E144D, E144I,E147R, E147Q, E147A, I148D, I148E, I148V, E150R, E150A, E151R, T152V,T152A, T152H, T152Y, T152R, T152Q, A153R, A153Q, A153G, A153S, R154S,R154P, R154E, R154Q, R154T, K155G, K155E, K155P, K155W, K155D, K155Q,K155T, Y156V, Y156R, Y156E, Y156F, Y156D, Y156Q, Y156T, Y156M, N157E,N157D, N157R, N157P, F158L, F158V, F158R, F158N, F158E, F158T, Q159L,Q159G, Q159R, Q159W, Q159E, Q159T, P160L, P160I, P160S, P160A, P160R,P160F, P160E, P160T, P160M, V161W, V161E, V161I, V161Y, V179R, V179I,S187R, S187Q, S187A, S190D, S190E, S190Y, S191L, S191A, S191H, S191Y,S191R, S191W, S191E, S191Q, S191T, K193L, K193I, K193Y, K193R, K193F,K193E, K193Q, K194L, K194D, K194A, K194R, K194E, K194Q, Y195A, Y195Q,Y195H, N197L, N197D, N197S, N197R, N197P, N197E, N197Q, P200L, P200D,P200R, P200E, P200M, N201R, N201E, W203L, W203A, W203H, W203Y, W203R,W203F, D204R, D204N, D204S, R205L, R205Q, G216R, G216N, G216T, S218T,Y219R, Y219G, D220E, D220R, D220Q, D220T, K221I, K221V, K221R, K221E,K221Q, K221T, V222I, E224Q, E224Y, E224M, D228E, D228S, K229A, K229R,K229N, K229E, K229Q, R231L, R231A, R231E, R231F, G232R, G232E, G232P,E235R, E235W, K238S, K238R, K238W, K238E, K238Q, R239S, E240V, E240H,E240Y, E240N, E240D, E240T, E240M, D241R, D241N, D241Q, D241P, R258L,R258E, E262L, E262R, E262Q, N287L, N287H, N287R, N287F, N287E, N287Q,D290R, D290E, D290Q, D290F, E291R, I292E, I292Q, E297L, E297R, E297H,S299A, S299Q, N300E, N300D, N300R, N300Q, K302L, K302I, K302R, K302P,K302E, K302Q, K302M, R303D, R303E, R303Q, E307R, E307Q, D313P, Y315W,K318D, K318N, K321E, K321D, K321R, K321S, D322T, S323D, S323G, K324D,K324H, K324P, K324W, K324E, R325D, R325S, R325A, R325G, R325W, R325E,R325Q, L326H, E327R, E327I, E327Y, L328D, L328Y, L328R, L328W, L328E,W331L, W331E, W331F, E343L, E343Y, E343N, E343D, E343Q, E343M, E345D,E345P, K348L, K348A, K348Y, K348R, K348W, K348E, K348Q, M349I, M349Y,M349R, M349N, M349W, M349E, M349Q, M349T, N352L, N352R, N352E, N352Q,N352M, K353L, K353A, K353H, K353R, K353E, K353Q, E356R, E356W, N357H,G361D, G361P, D363H, D363P, I364L, I364E, I364P, I364M, K365D, K365S,K365R, K365N, K365E, F366L, F366R, F366H, F366Y, Y368L, Y368R, Q369L,Q369R, Q369E, Q369D, Y370H, D373L, D373R, D373E, D373N, Y375W, Y375A,F376R, F376E, F376Q, D377S, D377A, D377H, D377R, D377N, E380L, E380R,E380D, E380N, K382L, K382D, K382R, K382N, K382E, K382Q, K384L, K384S,K384R, K384E, K384D, K384Q, K384T, N385P, N385T, D386H, R388E, E389R,E389D, K392L, K392H, K392Y, K392R, K392F, K392E, K393A, K393H, K393Y,K393R, K393W, K393E, K393Q, K393T, K396I, K396A, K396R, K396E, K396Q,K396M, R397L, R397I, R397S, R397H, R397E, R397Q, E400L, E400R, E400Q,E400Y, S403L, S403A, S403Y, S403R, S403F, S403W, S403E, S403T, N407L,N407D, N407I, N407H, N407Y, N407R, N407F, N407W, N407E, N407Q, L408G,L408R, L408N, L408W, L408D, or L408T; or combinations thereof.

In one aspect, the disclosure provides polynucleotides encoding any ofthe polypeptides disclosed herein. The present disclosure also providesconstructs, vectors, plasmids that comprises the polynucleotides.

In another aspect, the disclosure provides a microorganism expressingany of the polypeptides disclosed herein. In some embodiments, thedisclosure provides a microorganism expressing a polypeptide comprisingan amino acid sequence that is at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-444. In some embodiments, themicroorganism is modified. In some embodiments, wherein the modifiedmicroorganism is genetically modified. In some embodiments, the modifiedmicroorganism is non-naturally occurring.

In some embodiments, the modified microorganism is derived fromEscherichia coli, (hereinafter referred to as E. coli), Corynebacterumglutamicum, Aspergillus oryzae, Pichia pastoris, Bacillus subtilis,Caldithrix abyssi, Anaerolinea thermophila, Thermoanaerobacteriumthermosaccharolyticum, Thermoanaerobacter thermohydrosulfuricus,Caldicellulosiruptor kronotskyensis, Dictyoglomus turgidum, Caldilineaaerophila, Rhodothermus marinus, Methanohalobium evestigatum,Clostridium cavendishii, Kosmotoga olearia, Butyricicoccuspullicaecorum, Clostridium thermobutyricum, Litorilinea aerophila,Enterobacter mori., Caldisericum exile, Dictyoglomus thermophilum,Rhodothermus profundi, Caldibacillus debilis., Caloramator quimbayensis,Methanosalsum zhilinae, Pseudothermotoga thermarum, Pseudothermotogahypogea, Pseudothermotoga lettingae, Geosporobacter subterraneus,Melioribacter roseus, Lysinibacillus sphaericus, Clostridiumstercorarium, Truepera radiovictrix, Thermoflexus hugenholtzii,Petrotoga mobilis, Spirochaeta thermophila, Thermofilum pendens,Thermoanaerobacter siderophilus, Thermoanaerobacter mathranii,Thermoanaerobacter italicus, Thermoanaerobacterium xylanolyticum,Thermoanaerobacterium saccharolyticum, Ignisphaera aggregans, Thermotogamaritima, Caldanaerobacter subterraneus, Mesotoga infera, Thermosinuscarboxydivorans, Halanaerobium congolense, Halanaerobiumsaccharolyticum, Gracilibacillus halophilus, Caldicoprobacter faecalis,Thermoanaerobacter uzonensis, Lactobacillus ingluviei, Petrotogamexicana, Defluviitoga tunisiensis, Petrotoga miotherma, Petrotogaolearia, Thermophagus xiamenensis, Treponema caldarium, Thermofilumuzonense.

In some embodiments, the polypeptides disclosed herein or microorganismexpressing the polypeptide is immobilized. In some embodiments, thepolypeptide or the microorganism expressing the polypeptide isimmobilized to a carrier or support. In some embodiments, the carrier orsupport is an organic composition. In some embodiments, the carrier orsupport is an inorganic composition.

In another aspect, the disclosure provides a method of producingtagatose, the method comprising: (a) contacting fructose with any of thepolypeptide disclosed herein or a microorganism expressing thepolypeptide; and (b) converting fructose to tagatose. In someembodiments, the method comprises: (a) contacting fructose with apolypeptide or a microorganism expressing the polypeptide, wherein thepolypeptide comprises an amino acid sequence that is at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1-444; and (b)converting fructose to tagatose. In some embodiments, step (b) convertsfructose to tagatose through C4-epimerization of fructose. In someembodiments, the polypeptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-444. In some embodiments, thepolypeptide consists essentially of an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 1-444. In some embodiments, thepolypeptide consists of an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1-444. In some embodiments, the polypeptide iscapable of converting fructose to tagatose through C4-epimerization offructose. In some embodiments, the polypeptide has D-fructoseC4-epimerase activity.

A method for producing a tagatose composition, comprising the steps of:(a) providing a starting composition comprising greater than about 0.3%,about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%, about 90% of fructose by weight; (b) contacting the startingcomposition with any of the polypeptide disclosed herein or amicroorganism expressing the polypeptide; and (c) producing a tagatosecomposition comprising tagatose. In some embodiments, the polypeptide iscapable of converting fructose to tagatose through epimerization at thecarbon-4 position of fructose.

In one aspect, the disclosure provides a method for producing a tagatosecomposition, comprising the steps of: (a) providing a startingcomposition comprising greater than about 0.3%, about 0.5%, about 1%,about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90% offructose by weight; (b) contacting the starting composition with any ofthe polypeptide disclosed herein or a microorganism expressing thepolypeptide; and (c) producing a tagatose composition comprisingtagatose. In some embodiments, the polypeptide is capable of convertingfructose to tagatose through epimerization at the carbon-4 position offructose.

In some embodiments, the tagatose composition comprises tagatose in anamount greater than about 0.1%, about 0.2%, about 0.3%, about 0.5%,about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, or about 40% by weight. In some embodiments, contactingfructose with the polypeptide or the microorganism expressing thepolypeptide is performed at a temperature between about 40° C. and about100° C. In some embodiments, the contacting of fructose with thepolypeptide or the microorganism expressing the polypeptide is performedat between about pH 4.5 and about pH 8. In some embodiments, thecontacting of fructose with the polypeptide or the microorganismexpressing the polypeptide is performed in the presence of a metal ion.

In some embodiments, the tagatose product is separated from the feedstock (fructose, glucose, and/or sucrose, etc.) through chromatographicmeans. In some embodiments, the isolated tagatose is further purifiedwith crystallization.

DESCRIPTION OF THE DRAWINGS

The invention can be more completely understood with reference to thefollowing drawings.

FIG. 1 depicts the measured fructose to tagatose yield for twenty-threethermophilic D-fructose C4-epimerases (Scaffold1). The FC4Es werereacted with 20 mM fructose at 60° C. overnight.

FIG. 2 depicts the measured fructose to tagatose yield for fifty-threethermophilic D-fructose C4-epimerases (Scaffold2). The FC4Es werereacted with 20 mM fructose at 60° C. overnight.

FIG. 3 depicts measured Q-tof traces of the conversion of fructose (20mM) to tagatose for 8 of the thermophilic D-fructose C4-epimerases(Scaffold 1).

FIG. 4 depicts measured Q-tof traces of the conversion of fructose (20mM) to tagatose for 8 of the thermophilic D-fructose C4-epimerases(Scaffold 1).

FIG. 5 depicts the impact of metal salt on measured tagatose yield fromfructose for 9 FC4Es.

FIG. 6 depicts the relative fructose to tagatose yield when a highconcentration of fructose substrate (1M) was used in the reaction.

FIG. 7 depicts the relative fructose to tagatose yield for 8 FC4Es afterthe FC4Es were incubated at 60° C. for 24 hours prior to reacting withfructose.

FIG. 8 depicts the impact of temperature on tagatose yield for 3 FC4Es.

FIG. 9 depicts the fructose to tagatose yield over time for 4 FC4Es.

FIG. 10 depicts the effect of protein concentration on tagatose yieldfor 4 FC4Es

FIG. 11 depicts the impact of fructose concentration on tagatose yieldfor the FC4E, pA06238.

FIG. 12 depicts the effect of preincubating pA06238 at 80° C. fordifferent amounts of time on tagatose yield.

FIG. 13 depicts the fold change in expression and activity for 30 DNAoptimized variants of pA06238.

FIG. 14 depicts the fructose to tagatose yield of pA06238 immobilized on22 commercially available resins.

FIG. 15 depicts the percent tagatose converted from fructose over timefrom a fixed bed reactor packed with immobilized pA06238.

FIG. 16 depicts the fold change in fructose to tagatose conversion foractive site mutants of pA06234.

FIG. 17 depicts a sequence alignment of the native thermophilic FC4Es(SEQ ID NOs: 1-23 and SEQ ID NOs: 321-373). The residues are numberedbased on the pA06238 sequence (SEQ ID NO: 6). Black squares at the topof the alignment show residue positions that were mutated in Example 12(Active Site Mutant), Example 13 (Stability Expression Mutant), andExample 14 (Lys/Cys Only Mutant and Lys/Cys Mutant)

FIG. 18 depicts the fold change in fructose to tagatose conversion formembers of the FC4E active site mutant library (reaction time 1 hour).

FIG. 19 depicts the fold change in fructose to tagatose conversion formembers of the FC4E active site mutant library (reaction time 24 hours).

FIG. 20 depicts the sequence alignment of all computationally designedstability/expression mutants from Example 13 (SEQ ID NOs 46-136). Onlyresidue positions that were mutated in at least one mutant FC4E areshown. Amino acids that were not mutated are shown as a “.”.

FIG. 21 depicts the fold change in total conversion (amount of expressedprotein multiplied by the tagatose yield from a standard amount ofenzyme) for the FC4E computational designs.

FIG. 22 depicts the sequence alignment of all lysine/cysteine onlymutants from Example 14 (SEQ ID NOs 137-230). Only residue positionsthat were mutated in at least one mutant FC4E are shown. Amino acidsthat were not mutated are shown as a “.”.

FIG. 23 depicts the sequence alignment of all lysine/cysteine mutantsfrom Example 14 that allowed surrounding residues to mutate (SEQ ID NOs232-320). Only residue positions that were mutated in at least onemutant FC4E are shown. Amino acids that were not mutated are shown as a“.”.

FIG. 24 depicts the fold change in tagatose yield for a subset of theFC4E computational designs from Example 14.

DETAILED DESCRIPTION I. Definitions

Hereinafter, the present invention will be described in more detailbased on specific embodiments and examples. However, the scope the ofthe disclosure is not limited to these embodiments and examples.Descriptions of details apparent to those skilled in the art havingordinary knowledge in this technical field or relevant field will beomitted herein.

As used herein, the term CN refers to a carbon position defined inaccordance with IUPAC nomenclature, wherein N is an integer of 1 ormore. Specifically, “epimerization at the carbon 4 position” isexpressed as “C4-epimerization.”

As used herein, fructose refers to the monosaccharide D-fructose andtagatose refers to the monosaccharide D-tagatose.

The term “percent identity,” as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, New York (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, New York (1993); Computer Analysis of Sequence Data,Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NewJersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G.,ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M.and Devereux, J., eds.) Stockton Press, New York (1991). Preferredmethods to determine identity are designed to give the best matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing sequence analysis software such as the Megalign program of theLASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of the sequences may be performed using the Clustalmethod of alignment (Higgins et al., CABIOS. 5:151 (1989)) with thedefault parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the Clustal method may beselected: KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The term “modification” of an amino acid residue refers to substitutionof a different amino acid residue for an original amino acid residue,deletion of an original amino acid residue, or addition of an extraamino acid residue. The “modification” preferably refers to substitutionof a different amino acid residue for an original amino acid residue.Specifically, in the present invention, “modification of the charge ofan amino acid residue” preferably refers to amino acid substitutions.

II. Polypeptides

In one aspect, the disclosure provides a polypeptide comprising an aminoacid sequence having at least 60% sequence identity to any one of SEQ IDNOs:1-444, wherein the polypeptide has D-fructose C4-epimerase activity.In further embodiments, the polypeptide comprises an amino acid sequencethat is at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.5%, or at least 99.9% identical to any one of SEQ ID NOs: 1-444,wherein the polypeptide has D-fructose C4-epimerase activity. In someembodiments, the polypeptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-444. In some embodiments, thepolypeptide is capable of converting fructose to tagatose throughepimerization at carbon-4 position of fructose.

In some embodiments, the polypeptide consists essentially of an aminoacid sequence that is at least 65% identical to any one of SEQ ID NOs:1-444, wherein the polypeptide has D-fructose C4-epimerase activity. Insome embodiments, the polypeptide consists essentially of an amino acidsequence that is at least 70% identical to any one of SEQ ID NOs: 1-444,wherein the polypeptide has D-fructose C4-epimerase activity. In someembodiments, the polypeptide consists essentially of an amino acidsequence that is at least 75% identical to any one of SEQ ID NOs: 1-444,wherein the polypeptide has D-fructose C4-epimerase activity. In someembodiments, the polypeptide consists essentially of an amino acidsequence that is at least 80% identical to any one of SEQ ID NOs: 1-444,wherein the polypeptide has D-fructose C4-epimerase activity. In someembodiments, the polypeptide consists essentially of an amino acidsequence that is at least 85% identical to any one of SEQ ID NOs: 1-444,wherein the polypeptide has D-fructose C4-epimerase activity. In someembodiments, the polypeptide consists essentially of an amino acidsequence that is at least 90% identical to any one of SEQ ID NOs: 1-444,wherein the polypeptide has D-fructose C4-epimerase activity. In someembodiments, the polypeptide consists essentially of an amino acidsequence that is at least 95% identical to any one of SEQ ID NOs: 1-444,wherein the polypeptide has D-fructose C4-epimerase activity. In someembodiments, the polypeptide consists essentially of an amino acidsequence that is at least 98% identical to any one of SEQ ID NOs: 1-444,wherein the polypeptide has D-fructose C4-epimerase activity. In someembodiments, the polypeptide consists essentially of an amino acidsequence that is at least 99% identical to any one of SEQ ID NOs: 1-444,wherein the polypeptide has D-fructose C4-epimerase activity.

In other embodiments, the polypeptide comprises one or moremodifications at the amino acid residue corresponding to position L3,S4, K5, D6, Y7, L8, R9, K10, K11, V13, Y14, S15, I16, C17, S18, S19,S21, Y22, E25, S27, E29, F30, K32, E33, K34, G35, D36, Y37, E41, T43,P44, H45, Q46, Q49, F50, Y53, S54, M56, E59, K62, N63, F64, M66, K67,K70, E71, K72, E75, E76, D77, K78, I80, D84, H85, L89, Q92, D93, E94,S96, P97, T98, N101, K102, K104, D105, R108, F110, S113, Y115, K116,K117, H119, D121, C122, S123, M124, P125, L126, S127, D128, D129, P130,K131, V132, P134, Y135, E136, K137, E140, R141, T142, R143, E144, F146,E147, I148, E150, E151, T152, A153, R154, K155, Y156, N157, F158, Q159,P160, V161, T166, D167, V168, P169, I170, A171, G172, G173, G174, E175,E176, E177, G178, V179, V182, D184, S187, A188, I189, S190, S191, L192,K193, K194, Y195, N197, D198, V199, P200, N201, I202, W203, D204, R205,G208, V210, I211, M212, L213, I215, G216, F217, S218, Y219, D220, K221,V222, E224, D228, K229, V230, R231, G232, I233, L234, E235, V237, K238,R239, E240, D241, L242, V244, E245, G246, H247, S248, T249, D250, A253,Y255, A256, R258, N259, E262, R266, K269, V270, G271, P272, A273, R280,G281, V282, L284, S286, N287, D290, E291, I292, E295, R296, E297, S299,N300, K302, R303, E307, D313, Y315, K318, Y319, K321, D322, S323, K324,R325, L326, E327, L328, I330, W331, N333, L334, L335, D336, R337, R339,Y340, E343, Y344, E345, K348, M349, V350, N352, K353, E356, N357, F358,E360, G361, V362, D363, I364, K365, F366, Y368, Q369, Y370, Y372, D373,S374, Y375, F376, D377, R379, E380, K382, M383, K384, N385, D386, R388,E389, K392, K393, K396, R397, E400, S403, N407, or L408 of SEQ ID NO:6;or combinations thereof.

In further embodiments, the polypeptide comprises one or more ofmodifications: (1) the amino acid residue corresponding to position L3of SEQ ID NO:6 is GLU, or THR; (2) the amino acid residue correspondingto position S4 of SEQ ID NO:6 is ARG, ASN, ASP, GLN, GLU, LEU, MET, orPRO; (3) the amino acid residue corresponding to position K5 of SEQ IDNO:6 is ALA, ARG, GLN, HIS, LEU, MET, PHE, TRP, or VAL; (4) the aminoacid residue corresponding to position D6 of SEQ ID NO:6 is ALA, ARG,ASN, GLN, GLU, HIS, or LEU; (5) the amino acid residue corresponding toposition Y7 of SEQ ID NO:6 is ASN, HIS, or TRP; (6) the amino acidresidue corresponding to position L8 of SEQ ID NO:6 is ASN, or GLY; (7)the amino acid residue corresponding to position R9 of SEQ ID NO:6 isGLN, GLU, HIS, ILE, LEU, or THR; (8) the amino acid residuecorresponding to position K10 of SEQ ID NO:6 is ALA, ARG, ASN, ASP, GLU,LEU, MET, PRO, or VAL; (9) the amino acid residue corresponding toposition K11 of SEQ ID NO:6 is ARG, GLN, GLU, HIS, ILE, LEU, PHE, TRP,TYR, or VAL; (10) the amino acid residue corresponding to position V13of SEQ ID NO:6 is ILE; (11) the amino acid residue corresponding toposition Y14 of SEQ ID NO:6 is PHE; (12) the amino acid residuecorresponding to position S15 of SEQ ID NO:6 is ALA; (13) the amino acidresidue corresponding to position I16 of SEQ ID NO:6 is VAL; (14) theamino acid residue corresponding to position C17 of SEQ ID NO:6 is ALA,ARG, GLY, ILE, LEU, SER, THR, or VAL; (15) the amino acid residuecorresponding to position S18 of SEQ ID NO:6 is ALA, or ASN; (16) theamino acid residue corresponding to position S19 of SEQ ID NO:6 is ASN;(17) the amino acid residue corresponding to position S21 of SEQ ID NO:6is ARG, GLN, GLU, LYS, or PRO; (18) the amino acid residue correspondingto position Y22 of SEQ ID NO:6 is TRP; (19) the amino acid residuecorresponding to position E25 of SEQ ID NO:6 is ARG, or ILE; (20) theamino acid residue corresponding to position S27 of SEQ ID NO:6 is ALA,or CYS; (21) the amino acid residue corresponding to position E29 of SEQID NO:6 is ARG, GLN, ILE, or LEU; (22) the amino acid residuecorresponding to position F30 of SEQ ID NO:6 is ALA, HIS, or THR; (23)the amino acid residue corresponding to position K32 of SEQ ID NO:6 isALA, ARG, GLN, ILE, LEU, MET, or SER; (24) the amino acid residuecorresponding to position E33 of SEQ ID NO:6 is ARG, ASN, ASP, GLN, HIS,or THR; (25) the amino acid residue corresponding to position K34 of SEQID NO:6 is ARG, ASP, GLN, GLU, HIS, ILE, LEU, MET, PHE, SER, THR, TYR,or VAL; (26) the amino acid residue corresponding to position G35 of SEQID NO:6 is ASN, ASP, HIS, or SER; (27) the amino acid residuecorresponding to position D36 of SEQ ID NO:6 is ARG, ASN, GLN, GLU, HIS,LEU, PHE, SER, THR, TRP, or TYR; (28) the amino acid residuecorresponding to position Y37 of SEQ ID NO:6 is ARG, ASP, GLU, or PRO;(29) the amino acid residue corresponding to position E41 of SEQ ID NO:6is ALA, or GLN; (30) the amino acid residue corresponding to positionH45 of SEQ ID NO:6 is ALA, GLN, LYS, or PHE; (31) the amino acid residuecorresponding to position Q46 of SEQ ID NO:6 is ALA, ARG, SER, THR, orTRP; (32) the amino acid residue corresponding to position Q49 of SEQ IDNO:6 is HIS, ILE, PHE, or TRP; (33) the amino acid residue correspondingto position F50 of SEQ ID NO:6 is ARG, GLN, HIS, or SER; (34) the aminoacid residue corresponding to position Y53 of SEQ ID NO:6 is ALA, PHE,SER, or VAL; (35) the amino acid residue corresponding to position S54of SEQ ID NO:6 is THR; (36) the amino acid residue corresponding toposition M56 of SEQ ID NO:6 is TRP; (37) the amino acid residuecorresponding to position E59 of SEQ ID NO:6 is ARG, SER, or THR; (38)the amino acid residue corresponding to position K62 of SEQ ID NO:6 isALA, ARG, TYR, or VAL; (39) the amino acid residue corresponding toposition N63 of SEQ ID NO:6 is ARG, ASP, GLN, or GLU; (40) the aminoacid residue corresponding to position F64 of SEQ ID NO:6 is GLU, orLEU; (41) the amino acid residue corresponding to position M66 of SEQ IDNO:6 is GLU, PHE, or TYR; (42) the amino acid residue corresponding toposition K67 of SEQ ID NO:6 is ARG, ASP, GLN, GLU, SER, or THR; (43) theamino acid residue corresponding to position K70 of SEQ ID NO:6 is ARG,ASP, GLN, or LEU; (44) the amino acid residue corresponding to positionE71 of SEQ ID NO:6 is ARG, GLN, or SER; (45) the amino acid residuecorresponding to position K72 of SEQ ID NO:6 is ARG, GLN, GLU, HIS, ILE,LEU, THR, or VAL; (46) the amino acid residue corresponding to positionE75 of SEQ ID NO:6 is ARG, ASP, PRO, or SER; (47) the amino acid residuecorresponding to position E76 of SEQ ID NO:6 is ARG, LEU, or PRO; (48)the amino acid residue corresponding to position D77 of SEQ ID NO:6 isARG, ASN, or SER; (49) the amino acid residue corresponding to positionK78 of SEQ ID NO:6 is ARG, ASP, GLN, LEU, or MET; (50) the amino acidresidue corresponding to position I80 of SEQ ID NO:6 is ARG, GLN, orLEU; (51) the amino acid residue corresponding to position D84 of SEQ IDNO:6 is ALA, ASN, GLY, or PRO; (52) the amino acid residue correspondingto position H85 of SEQ ID NO:6 is ALA; (53) the amino acid residuecorresponding to position L89 of SEQ ID NO:6 is GLY, or SER; (54) theamino acid residue corresponding to position Q92 of SEQ ID NO:6 is ALA,ASP, GLY, MET, or PRO; (55) the amino acid residue corresponding toposition D93 of SEQ ID NO:6 is HIS; (56) the amino acid residuecorresponding to position E94 of SEQ ID NO:6 is ARG, GLN, LEU, or TYR;(57) the amino acid residue corresponding to position S96 of SEQ ID NO:6is ALA, ARG, or GLU; (58) the amino acid residue corresponding toposition P97 of SEQ ID NO:6 is ASP, or GLU; (59) the amino acid residuecorresponding to position T98 of SEQ ID NO:6 is ARG, GLN, GLU, ILE, TRP,or VAL; (60) the amino acid residue corresponding to position N101 ofSEQ ID NO:6 is ALA, ARG, ASP, GLN, GLU, HIS, LYS, SER, or THR; (61) theamino acid residue corresponding to position K102 of SEQ ID NO:6 is ARG,GLN, GLU, LEU, TRP, or TYR; (62) the amino acid residue corresponding toposition K104 of SEQ ID NO:6 is ARG, ASN, GLN, GLU, ILE, PHE, or VAL;(63) the amino acid residue corresponding to position D105 of SEQ IDNO:6 is GLN, GLU, or ILE; (64) the amino acid residue corresponding toposition R108 of SEQ ID NO:6 is ILE; (65) the amino acid residuecorresponding to position F110 of SEQ ID NO:6 is TYR; (66) the aminoacid residue corresponding to position S113 of SEQ ID NO:6 is ALA; (67)the amino acid residue corresponding to position Y115 of SEQ ID NO:6 isPHE; (68) the amino acid residue corresponding to position K116 of SEQID NO:6 is ARG, ASP, GLN, GLU, HIS, ILE, LEU, SER, THR, TRP, or TYR;(69) the amino acid residue corresponding to position K117 of SEQ IDNO:6 is ARG, LEU, MET, TRP, or VAL; (70) the amino acid residuecorresponding to position H119 of SEQ ID NO:6 is ALA, ASN, GLY, PRO, orSER; (71) the amino acid residue corresponding to position D121 of SEQID NO:6 is ALA, or ASN; (72) the amino acid residue corresponding toposition C122 of SEQ ID NO:6 is ALA, ARG, GLU, GLY, LYS, THR, or VAL;(73) the amino acid residue corresponding to position S123 of SEQ IDNO:6 is ALA, ARG, MET, or THR; (74) the amino acid residue correspondingto position M124 of SEQ ID NO:6 is ARG, or LYS; (75) the amino acidresidue corresponding to position P125 of SEQ ID NO:6 is ALA; (76) theamino acid residue corresponding to position L126 of SEQ ID NO:6 is CYS;(77) the amino acid residue corresponding to position S127 of SEQ IDNO:6 is ALA; (78) the amino acid residue corresponding to position D128of SEQ ID NO:6 is GLY; (79) the amino acid residue corresponding toposition D129 of SEQ ID NO:6 is ARG, GLN, GLU, LEU, or TRP; (80) theamino acid residue corresponding to position P130 of SEQ ID NO:6 is SER;(81) the amino acid residue corresponding to position K131 of SEQ IDNO:6 is ALA, ARG, ASP, GLN, GLU, LEU, SER, or TYR; (82) the amino acidresidue corresponding to position V132 of SEQ ID NO:6 is ARG, GLU, SER,THR, or TYR; (83) the amino acid residue corresponding to position P134of SEQ ID NO:6 is ALA, ASP, or HIS; (84) the amino acid residuecorresponding to position Y135 of SEQ ID NO:6 is ARG, ASN, ASP, GLN,LEU, LYS, MET, PRO, THR, or TRP; (85) the amino acid residuecorresponding to position E136 of SEQ ID NO:6 is ARG, ASP, GLN, PRO,SER, THR, or TRP; (86) the amino acid residue corresponding to positionK137 of SEQ ID NO:6 is ARG, GLN, GLU, ILE, LEU, THR, TRP, or VAL; (87)the amino acid residue corresponding to position E140 of SEQ ID NO:6 isARG, ASP, ILE, or LEU; (88) the amino acid residue corresponding toposition R141 of SEQ ID NO:6 is ASP, GLN, HIS, LEU, TRP, or TYR; (89)the amino acid residue corresponding to position T142 of SEQ ID NO:6 isGLU; (90) the amino acid residue corresponding to position R143 of SEQID NO:6 is ALA, or VAL; (91) the amino acid residue corresponding toposition E144 of SEQ ID NO:6 is ARG, ASP, GLN, or ILE; (92) the aminoacid residue corresponding to position F146 of SEQ ID NO:6 is CYS; (93)the amino acid residue corresponding to position E147 of SEQ ID NO:6 isALA, ARG, GLN, or LYS; (94) the amino acid residue corresponding toposition I148 of SEQ ID NO:6 is ASP, GLU, or VAL; (95) the amino acidresidue corresponding to position E150 of SEQ ID NO:6 is ALA, or ARG;(96) the amino acid residue corresponding to position E151 of SEQ IDNO:6 is ARG; (97) the amino acid residue corresponding to position T152of SEQ ID NO:6 is ALA, ARG, GLN, HIS, TYR, or VAL; (98) the amino acidresidue corresponding to position A153 of SEQ ID NO:6 is ARG, GLN, GLY,or SER; (99) the amino acid residue corresponding to position R154 ofSEQ ID NO:6 is GLN, GLU, PRO, SER, or THR; (100) the amino acid residuecorresponding to position K155 of SEQ ID NO:6 is ARG, ASN, ASP, GLN,GLU, GLY, HIS, PRO, THR, TRP, or TYR; (101) the amino acid residuecorresponding to position Y156 of SEQ ID NO:6 is ARG, ASP, GLN, GLU,MET, PHE, THR, or VAL; (102) the amino acid residue corresponding toposition N157 of SEQ ID NO:6 is ARG, ASP, GLU, or PRO; (103) the aminoacid residue corresponding to position F158 of SEQ ID NO:6 is ARG, ASN,GLU, LEU, THR, or VAL; (104) the amino acid residue corresponding toposition Q159 of SEQ ID NO:6 is ARG, GLU, GLY, LEU, THR, or TRP; (105)the amino acid residue corresponding to position P160 of SEQ ID NO:6 isALA, ARG, GLU, ILE, LEU, MET, PHE, SER, or THR; (106) the amino acidresidue corresponding to position V161 of SEQ ID NO:6 is GLU, ILE, TRP,or TYR; (107) the amino acid residue corresponding to position T166 ofSEQ ID NO:6 is ILE, LEU, or MET; (108) the amino acid residuecorresponding to position D167 of SEQ ID NO:6 is ALA, ASN, GLN, THR, orVAL; (109) the amino acid residue corresponding to position V168 of SEQID NO:6 is ARG, or MET; (110) the amino acid residue corresponding toposition I170 of SEQ ID NO:6 is LYS, MET, or THR; (111) the amino acidresidue corresponding to position A171 of SEQ ID NO:6 is PRO; (112) theamino acid residue corresponding to position G173 of SEQ ID NO:6 is SER;(113) the amino acid residue corresponding to position E175 of SEQ IDNO:6 is ARG, LYS, or VAL; (114) the amino acid residue corresponding toposition E176 of SEQ ID NO:6 is PHE, or TYR; (115) the amino acidresidue corresponding to position G178 of SEQ ID NO:6 is PRO; (116) theamino acid residue corresponding to position V179 of SEQ ID NO:6 is ARG,or ILE; (117) the amino acid residue corresponding to position V182 ofSEQ ID NO:6 is ALA, LYS, or PRO; (118) the amino acid residuecorresponding to position D184 of SEQ ID NO:6 is GLU; (119) the aminoacid residue corresponding to position S187 of SEQ ID NO:6 is ALA, ARG,ASN, GLN, GLU, LYS, or THR; (120) the amino acid residue correspondingto position A188 of SEQ ID NO:6 is MET, PHE, or THR; (121) the aminoacid residue corresponding to position I189 of SEQ ID NO:6 is LEU; (122)the amino acid residue corresponding to position S190 of SEQ ID NO:6 isASP, GLU, or TYR; (123) the amino acid residue corresponding to positionS191 of SEQ ID NO:6 is ALA, ARG, GLN, GLU, HIS, ILE, LEU, THR, TRP, TYR,or VAL; (124) the amino acid residue corresponding to position L192 ofSEQ ID NO:6 is HIS, or PHE; (125) the amino acid residue correspondingto position K193 of SEQ ID NO:6 is ALA, ARG, GLN, GLU, ILE, LEU, PHE, orTYR; (126) the amino acid residue corresponding to position K194 of SEQID NO:6 is ALA, ARG, ASP, GLN, GLU, LEU, SER, or THR; (127) the aminoacid residue corresponding to position Y195 of SEQ ID NO:6 is ALA, GLN,or HIS; (128) the amino acid residue corresponding to position N197 ofSEQ ID NO:6 is ALA, ARG, ASP, GLN, GLU, LEU, LYS, PRO, or SER; (129) theamino acid residue corresponding to position D198 of SEQ ID NO:6 is LYS;(130) the amino acid residue corresponding to position V199 of SEQ IDNO:6 is CYS, HIS, ILE, or LEU; (131) the amino acid residuecorresponding to position P200 of SEQ ID NO:6 is ARG, ASP, GLU, GLY,LEU, or MET; (132) the amino acid residue corresponding to position N201of SEQ ID NO:6 is ARG, GLN, GLU, or LYS; (133) the amino acid residuecorresponding to position I202 of SEQ ID NO:6 is ALA, or MET; (134) theamino acid residue corresponding to position W203 of SEQ ID NO:6 is ALA,ARG, HIS, LEU, PHE, or TYR; (135) the amino acid residue correspondingto position D204 of SEQ ID NO:6 is ARG, ASN, or SER; (136) the aminoacid residue corresponding to position R205 of SEQ ID NO:6 is GLN, orLEU; (137) the amino acid residue corresponding to position G208 of SEQID NO:6 is ALA; (138) the amino acid residue corresponding to positionV210 of SEQ ID NO:6 is PRO, or THR; (139) the amino acid residuecorresponding to position I211 of SEQ ID NO:6 is VAL; (140) the aminoacid residue corresponding to position M212 of SEQ ID NO:6 is ARG, LEU,or LYS; (141) the amino acid residue corresponding to position L213 ofSEQ ID NO:6 is MET; (142) the amino acid residue corresponding toposition I215 of SEQ ID NO:6 is VAL; (143) the amino acid residuecorresponding to position G216 of SEQ ID NO:6 is ARG, ASN, or THR; (144)the amino acid residue corresponding to position S218 of SEQ ID NO:6 isGLY, or THR; (145) the amino acid residue corresponding to position Y219of SEQ ID NO:6 is ARG, ASP, GLY, or HIS; (146) the amino acid residuecorresponding to position D220 of SEQ ID NO:6 is ARG, GLN, GLU, or THR;(147) the amino acid residue corresponding to position K221 of SEQ IDNO:6 is ARG, GLN, GLU, ILE, THR, or VAL; (148) the amino acid residuecorresponding to position V222 of SEQ ID NO:6 is ILE; (149) the aminoacid residue corresponding to position E224 of SEQ ID NO:6 is GLN, MET,or TYR; (150) the amino acid residue corresponding to position D228 ofSEQ ID NO:6 is GLU, or SER; (151) the amino acid residue correspondingto position K229 of SEQ ID NO:6 is ALA, ARG, ASN, GLN, GLU, SER, or THR;(152) the amino acid residue corresponding to position V230 of SEQ IDNO:6 is ALA; (153) the amino acid residue corresponding to position R231of SEQ ID NO:6 is ALA, GLU, LEU, or PHE; (154) the amino acid residuecorresponding to position G232 of SEQ ID NO:6 is ARG, ASP, GLU, LYS, orPRO; (155) the amino acid residue corresponding to position I233 of SEQID NO:6 is LEU; (156) the amino acid residue corresponding to positionL234 of SEQ ID NO:6 is ALA, or MET; (157) the amino acid residuecorresponding to position E235 of SEQ ID NO:6 is ARG, or TRP; (158) theamino acid residue corresponding to position V237 of SEQ ID NO:6 is LEU;(159) the amino acid residue corresponding to position K238 of SEQ IDNO:6 is ARG, GLN, GLU, SER, or TRP; (160) the amino acid residuecorresponding to position R239 of SEQ ID NO:6 is SER; (161) the aminoacid residue corresponding to position E240 of SEQ ID NO:6 is ASN, ASP,HIS, MET, THR, TYR, or VAL; (162) the amino acid residue correspondingto position D241 of SEQ ID NO:6 is ARG, ASN, GLN, GLY, or PRO; (163) theamino acid residue corresponding to position L242 of SEQ ID NO:6 is TRP;(164) the amino acid residue corresponding to position V244 of SEQ IDNO:6 is LEU; (165) the amino acid residue corresponding to position E245of SEQ ID NO:6 is ALA, or GLN; (166) the amino acid residuecorresponding to position G246 of SEQ ID NO:6 is ALA, CYS, or VAL; (167)the amino acid residue corresponding to position H247 of SEQ ID NO:6 isALA; (168) the amino acid residue corresponding to position T249 of SEQID NO:6 is SER; (169) the amino acid residue corresponding to positionD250 of SEQ ID NO:6 is ALA, or ASN; (170) the amino acid residuecorresponding to position A253 of SEQ ID NO:6 is ASN, ASP, PRO, SER, orTHR; (171) the amino acid residue corresponding to position Y255 of SEQID NO:6 is ASP, GLN, GLU, or LYS; (172) the amino acid residuecorresponding to position A256 of SEQ ID NO:6 is LEU; (173) the aminoacid residue corresponding to position R258 of SEQ ID NO:6 is GLU, orLEU; (174) the amino acid residue corresponding to position N259 of SEQID NO:6 is ALA, ARG, ASP, GLU, or TRP; (175) the amino acid residuecorresponding to position E262 of SEQ ID NO:6 is ARG, GLN, or LEU; (176)the amino acid residue corresponding to position R266 of SEQ ID NO:6 isALA, or VAL; (177) the amino acid residue corresponding to position K269of SEQ ID NO:6 is ARG, ASN, ILE, LEU, or VAL; (178) the amino acidresidue corresponding to position G271 of SEQ ID NO:6 is ALA, or ASP;(179) the amino acid residue corresponding to position A273 of SEQ IDNO:6 is TRP; (180) the amino acid residue corresponding to position R280of SEQ ID NO:6 is GLU; (181) the amino acid residue corresponding toposition G281 of SEQ ID NO:6 is ALA; (182) the amino acid residuecorresponding to position V282 of SEQ ID NO:6 is ILE; (183) the aminoacid residue corresponding to position L284 of SEQ ID NO:6 is LYS; (184)the amino acid residue corresponding to position S286 of SEQ ID NO:6 isALA, ASP, or VAL; (185) the amino acid residue corresponding to positionN287 of SEQ ID NO:6 is ARG, ASP, GLN, GLU, HIS, LEU, or PHE; (186) theamino acid residue corresponding to position D290 of SEQ ID NO:6 is ALA,ARG, ASN, GLN, GLU, LYS, or PHE; (187) the amino acid residuecorresponding to position E291 of SEQ ID NO:6 is ARG, or ASP; (188) theamino acid residue corresponding to position I292 of SEQ ID NO:6 is GLN,GLU, or LEU; (189) the amino acid residue corresponding to position E295of SEQ ID NO:6 is PRO; (190) the amino acid residue corresponding toposition R296 of SEQ ID NO:6 is ASP, GLU, or TYR; (191) the amino acidresidue corresponding to position E297 of SEQ ID NO:6 is ARG, GLN, GLY,HIS, LEU, or LYS; (192) the amino acid residue corresponding to positionS299 of SEQ ID NO:6 is ALA, or GLN; (193) the amino acid residuecorresponding to position N300 of SEQ ID NO:6 is ARG, ASP, GLN, or GLU;(194) the amino acid residue corresponding to position K302 of SEQ IDNO:6 is ARG, GLN, GLU, ILE, LEU, MET, or PRO; (195) the amino acidresidue corresponding to position R303 of SEQ ID NO:6 is ASP, GLN, orGLU; (196) the amino acid residue corresponding to position E307 of SEQID NO:6 is ARG, or GLN; (197) the amino acid residue corresponding toposition D313 of SEQ ID NO:6 is PRO; (198) the amino acid residuecorresponding to position Y315 of SEQ ID NO:6 is TRP; (199) the aminoacid residue corresponding to position K318 of SEQ ID NO:6 is ARG, ASN,ASP, or GLU; (200) the amino acid residue corresponding to position K321of SEQ ID NO:6 is ARG, ASP, GLU, HIS, SER, or THR; (201) the amino acidresidue corresponding to position D322 of SEQ ID NO:6 is THR; (202) theamino acid residue corresponding to position S323 of SEQ ID NO:6 is ASP,or GLY; (203) the amino acid residue corresponding to position K324 ofSEQ ID NO:6 is ARG, ASP, GLU, HIS, PRO, SER, or TRP; (204) the aminoacid residue corresponding to position R325 of SEQ ID NO:6 is ALA, ASN,ASP, GLN, GLU, GLY, SER, or TRP; (205) the amino acid residuecorresponding to position L326 of SEQ ID NO:6 is HIS; (206) the aminoacid residue corresponding to position E327 of SEQ ID NO:6 is ARG, ILE,or TYR; (207) the amino acid residue corresponding to position L328 ofSEQ ID NO:6 is ARG, ASP, GLU, PHE, TRP, or TYR; (208) the amino acidresidue corresponding to position I330 of SEQ ID NO:6 is LEU; (209) theamino acid residue corresponding to position W331 of SEQ ID NO:6 is GLU,HIS, LEU, or PHE; (210) the amino acid residue corresponding to positionN333 of SEQ ID NO:6 is ALA, or SER; (211) the amino acid residuecorresponding to position L334 of SEQ ID NO:6 is TYR; (212) the aminoacid residue corresponding to position R339 of SEQ ID NO:6 is ALA, GLU,GLY, or THR; (213) the amino acid residue corresponding to position Y340of SEQ ID NO:6 is ALA, ARG, HIS, PHE, or PRO; (214) the amino acidresidue corresponding to position E343 of SEQ ID NO:6 is ASN, ASP, GLN,LEU, MET, or TYR; (215) the amino acid residue corresponding to positionY344 of SEQ ID NO:6 is HIS; (216) the amino acid residue correspondingto position E345 of SEQ ID NO:6 is ASP, or PRO; (217) the amino acidresidue corresponding to position K348 of SEQ ID NO:6 is ALA, ARG, GLN,GLU, LEU, TRP, TYR, or VAL; (218) the amino acid residue correspondingto position M349 of SEQ ID NO:6 is ALA, ARG, ASN, CYS, GLN, GLU, ILE,LYS, THR, TRP, or TYR; (219) the amino acid residue corresponding toposition V350 of SEQ ID NO:6 is ALA; (220) the amino acid residuecorresponding to position N352 of SEQ ID NO:6 is ARG, GLN, GLU, LEU, orMET; (221) the amino acid residue corresponding to position K353 of SEQID NO:6 is ALA, ARG, GLN, GLU, HIS, LEU, or TRP; (222) the amino acidresidue corresponding to position E356 of SEQ ID NO:6 is ARG, or TRP;(223) the amino acid residue corresponding to position N357 of SEQ IDNO:6 is HIS; (224) the amino acid residue corresponding to position F358of SEQ ID NO:6 is LEU; (225) the amino acid residue corresponding toposition E360 of SEQ ID NO:6 is ASP; (226) the amino acid residuecorresponding to position G361 of SEQ ID NO:6 is ASP, or PRO; (227) theamino acid residue corresponding to position V362 of SEQ ID NO:6 is ILE;(228) the amino acid residue corresponding to position D363 of SEQ IDNO:6 is HIS, or PRO; (229) the amino acid residue corresponding toposition I364 of SEQ ID NO:6 is GLU, LEU, MET, PRO, or TRP; (230) theamino acid residue corresponding to position K365 of SEQ ID NO:6 is ARG,ASN, ASP, GLU, HIS, SER, or THR; (231) the amino acid residuecorresponding to position F366 of SEQ ID NO:6 is ARG, HIS, LEU, or TYR;(232) the amino acid residue corresponding to position Y368 of SEQ IDNO:6 is ARG, or LEU; (233) the amino acid residue corresponding toposition Q369 of SEQ ID NO:6 is ARG, ASP, GLU, or LEU; (234) the aminoacid residue corresponding to position Y370 of SEQ ID NO:6 is HIS; (235)the amino acid residue corresponding to position Y372 of SEQ ID NO:6 isPRO; (236) the amino acid residue corresponding to position D373 of SEQID NO:6 is ALA, ARG, ASN, GLU, or LEU; (237) the amino acid residuecorresponding to position S374 of SEQ ID NO:6 is ALA, CYS, GLN, or THR;(238) the amino acid residue corresponding to position Y375 of SEQ IDNO:6 is ALA, or TRP; (239) the amino acid residue corresponding toposition F376 of SEQ ID NO:6 is ARG, GLN, GLU, HIS, or LYS; (240) theamino acid residue corresponding to position D377 of SEQ ID NO:6 is ALA,ARG, ASN, HIS, or SER; (241) the amino acid residue corresponding toposition R379 of SEQ ID NO:6 is CYS; (242) the amino acid residuecorresponding to position E380 of SEQ ID NO:6 is ARG, ASN, ASP, or LEU;(243) the amino acid residue corresponding to position K382 of SEQ IDNO:6 is ARG, ASN, ASP, GLN, GLU, or LEU; (244) the amino acid residuecorresponding to position M383 of SEQ ID NO:6 is LEU; (245) the aminoacid residue corresponding to position K384 of SEQ ID NO:6 is ARG, ASP,GLN, GLU, LEU, SER, or THR; (246) the amino acid residue correspondingto position N385 of SEQ ID NO:6 is PRO, or THR; (247) the amino acidresidue corresponding to position D386 of SEQ ID NO:6 is HIS; (248) theamino acid residue corresponding to position R388 of SEQ ID NO:6 is GLU;(249) the amino acid residue corresponding to position E389 of SEQ IDNO:6 is ARG, or ASP; (250) the amino acid residue corresponding toposition K392 of SEQ ID NO:6 is ARG, GLU, HIS, LEU, MET, PHE, or TYR;(251) the amino acid residue corresponding to position K393 of SEQ IDNO:6 is ALA, ARG, GLN, GLU, HIS, LEU, PHE, THR, TRP, or TYR; (252) theamino acid residue corresponding to position K396 of SEQ ID NO:6 is ALA,ARG, GLN, GLU, ILE, LEU, MET, or PHE; (253) the amino acid residuecorresponding to position R397 of SEQ ID NO:6 is GLN, GLU, HIS, ILE,LEU, LYS, or SER; (254) the amino acid residue corresponding to positionE400 of SEQ ID NO:6 is ARG, GLN, LEU, or TYR; (255) the amino acidresidue corresponding to position S403 of SEQ ID NO:6 is ALA, ARG, GLU,HIS, LEU, PHE, THR, TRP, or TYR; (256) the amino acid residuecorresponding to position N407 of SEQ ID NO:6 is ARG, ASP, GLN, GLU,HIS, ILE, LEU, PHE, TRP, or TYR; or (257) the amino acid residuecorresponding to position L408 of SEQ ID NO:6 is ARG, ASN, ASP, GLY,THR, or TRP; or combinations thereof.

In some embodiments, the polypeptide comprises one or more ofmodifications: L3E, L3T, S4M, S4D, S4R, S4L, S4Q, S4N, S4E, S4P, K5F,K5M, K5L, K5R, K5W, K5H, K5Q, K5V, K5A, D6R, D6L, D6H, D6Q, D6N, D6A,D6E, Y7W, Y7H, Y7N, L8N, L8G, R9L, R9T, R9H, R9Q, R9E, R9I, K10M, K10D,K10R, K10L, K10V, K10N, K10A, K10E, K10P, K11F, K11L, K11R, K11Y, K11W,K11V, K11Q, K11H, K11E, K11I, V13I, Y14F, S15A, I16V, C17S, C17L, C17R,C17T, C17G, C17V, C17A, C17I, S18N, S18A, S19N, S21K, S21R, S21Q, S21E,S21P, Y22W, E25R, E25I, S27C, S27A, E29Q, E29R, E29I, E29L, F30H, F30T,F30A, K32S, K32M, K32L, K32R, K32Q, K32A, K32I, E33R, E33T, E33H, E33Q,E33N, E33D, K34F, K34S, K34M, K34R, K34L, K34Y, K34T, K34H, K34Q, K34V,K34E, K34D, K34I, G35D, G35N, G35H, G35S, D36F, D36S, D36R, D36L, D36Y,D36T, D36W, D36H, D36Q, D36N, D36E, Y37D, Y37P, Y37R, Y37E, E41Q, E41A,H45F, H45K, H45Q, H45A, Q46S, Q46R, Q46T, Q46W, Q46A, Q49F, Q49H, Q49I,Q49W, F50Q, F50H, F50R, F50S, Y53F, Y53V, Y53S, Y53A, S54T, M56W, E59R,E59S, E59T, K62V, K62R, K62Y, K62A, N63E, N63D, N63R, N63Q, F64E, F64L,M66F, M66E, M66Y, K67S, K67R, K67T, K67Q, K67E, K67D, K70D, K70Q, K70R,K70L, E71Q, E71R, E71S, K72L, K72R, K72T, K72V, K72Q, K72H, K72E, K72I,E75D, E75P, E75R, E75S, E76P, E76L, E76R, D77N, D77R, D77S, K78M, K78R,K78L, K78Q, K78D, I80Q, I80R, I80L, D84P, D84N, D84G, D84A, H85A, L89S,L89G, Q92M, Q92D, Q92G, Q92A, Q92P, D93H, E94Q, E94R, E94Y, E94L, S96E,S96R, S96A, P97E, P97D, T98R, T98W, T98V, T98Q, T98E, T98I, N101S,N101D, N101R, N101T, N101H, N101Q, N101A, N101E, N101K, K102L, K102R,K102Y, K102W, K102Q, K102E, K104F, K104R, K104V, K104Q, K104N, K104E,K104I, D105E, D105Q, D105I, R108I, F110Y, S113A, Y115F, K116S, K116R,K116L, K116Y, K116T, K116W, K116H, K116Q, K116E, K116D, K116I, K117M,K117L, K117R, K117W, K117V, H119S, H119G, H119N, H119A, H119P, D121N,D121A, C122R, C122G, C122T, C122V, C122A, C122E, C122K, S123M, S123R,S123T, S123A, M124K, M124R, P125A, L126C, S127A, D128G, D129R, D129L,D129W, D129Q, D129E, P130S, K131S, K131R, K131L, K131Y, K131Q, K131A,K131E, K131D, V132S, V132R, V132Y, V132T, V132E, P134D, P134H, P134A,Y135K, Y135M, Y135D, Y135L, Y135R, Y135T, Y135W, Y135Q, Y135N, Y135P,E136S, E136R, E136P, E136W, E136T, E136Q, E136D, K137L, K137R, K137T,K137W, K137V, K137Q, K137E, K137I, E140D, E140R, E140I, E140L, R141L,R141Y, R141W, R141H, R141Q, R141D, T142E, R143V, R143A, E144Q, E144D,E144R, E144I, F146C, E147Q, E147K, E147R, E147A, I148E, I148V, I148D,E150R, E150A, E151R, T152R, T152Y, T152V, T152H, T152Q, T152A, A153Q,A153R, A153G, A153S, R154S, R154T, R154Q, R154E, R154P, K155R, K155Y,K155P, K155T, K155H, K155N, K155Q, K155G, K155W, K155E, K155D, Y156F,Y156M, Y156R, Y156T, Y156V, Y156Q, Y156E, Y156D, N157E, N157D, N157R,N157P, F158R, F158L, F158T, F158V, F158N, F158E, Q159R, Q159L, Q159G,Q159T, Q159W, Q159E, P160F, P160S, P160M, P160L, P160R, P160T, P160A,P160E, P160I, V161E, V161I, V161Y, V161W, T166M, T166L, T166I, D167T,D167V, D167Q, D167N, D167A, V168M, V168R, I170M, I170K, I170T, A171P,G173S, E175K, E175V, E175R, E176F, E176Y, G178P, V179R, V179I, V182P,V182K, V182A, D184E, S187R, S187T, S187N, S187Q, S187A, S187E, S187K,A188M, A188F, A188T, I189L, S190E, S190D, S190Y, S191L, S191R, S191Y,S191T, S191W, S191V, S191H, S191Q, S191A, S191E, S191I, L192F, L192H,K193F, K193R, K193L, K193Y, K193Q, K193A, K193E, K193I, K194S, K194R,K194L, K194T, K194Q, K194A, K194E, K194D, Y195H, Y195Q, Y195A, N197S,N197D, N197R, N197L, N197P, N197Q, N197A, N197E, N197K, D198K, V199H,V199L, V199I, V199C, P200M, P200L, P200R, P200G, P200E, P200D, N201Q,N201K, N201R, N201E, I202M, I202A, W203F, W203R, W203L, W203Y, W203H,W203A, D204N, D204R, D204S, R205Q, R205L, G208A, V210P, V210T, I211V,M212K, M212L, M212R, L213M, I215V, G216N, G216R, G216T, S218T, S218G,Y219D, Y219H, Y219R, Y219G, D220Q, D220R, D220E, D220T, K221R, K221T,K221V, K221Q, K221E, K221I, V222I, E224M, E224Q, E224Y, D228E, D228S,K229S, K229R, K229T, K229N, K229Q, K229A, K229E, V230A, R231E, R231L,R231F, R231A, G232D, G232R, G232P, G232E, G232K, I233L, L234M, L234A,E235W, E235R, V237L, K238S, K238R, K238W, K238Q, K238E, R239S, E240M,E240Y, E240T, E240V, E240N, E240H, E240D, D241R, D241G, D241Q, D241N,D241P, L242W, V244L, E245Q, E245A, G246C, G246V, G246A, H247A, T249S,D250N, D250A, A253S, A253P, A253T, A253N, A253D, Y255E, Y255D, Y255Q,Y255K, A256L, R258E, R258L, N259R, N259W, N259A, N259E, N259D, E262Q,E262R, E262L, R266V, R266A, K269L, K269R, K269V, K269N, K269I, G271D,G271A, A273W, R280E, G281A, V282I, L284K, S286D, S286V, S286A, N287F,N287R, N287L, N287H, N287Q, N287E, N287D, D290F, D290R, D290Q, D290N,D290A, D290E, D290K, E291D, E291R, I292Q, I292L, I292E, E295P, R296E,R296D, R296Y, E297R, E297L, E297G, E297H, E297Q, E297K, S299Q, S299A,N300E, N300D, N300Q, N300R, K302M, K302L, K302R, K302Q, K302E, K302P,K302I, R303E, R303D, R303Q, E307Q, E307R, D313P, Y315W, K318E, K318D,K318R, K318N, K321S, K321R, K321T, K321H, K321E, K321D, D322T, S323D,S323G, K324S, K324R, K324P, K324W, K324H, K324E, K324D, R325S, R325G,R325W, R325N, R325Q, R325A, R325E, R325D, L326H, E327R, E327I, E327Y,L328F, L328R, L328Y, L328W, L328E, L328D, I330L, W331E, W331H, W331L,W331F, N333A, N333S, L334Y, R339E, R339T, R339A, R339G, Y340F, Y340R,Y340H, Y340A, Y340P, E343M, E343L, E343Y, E343Q, E343N, E343D, Y344H,E345D, E345P, K348R, K348L, K348Y, K348W, K348V, K348Q, K348A, K348E,M349R, M349Y, M349C, M349T, M349W, M349N, M349Q, M349A, M349E, M349K,M349I, V350A, N352M, N352R, N352L, N352Q, N352E, K353R, K353L, K353W,K353H, K353Q, K353A, K353E, E356W, E356R, N357H, F358L, E360D, G361D,G361P, V362I, D363P, D363H, I364M, I364L, I364W, I364E, I364P, K365S,K365R, K365T, K365H, K365N, K365E, K365D, F366H, F366L, F366R, F366Y,Y368R, Y368L, Q369E, Q369D, Q369R, Q369L, Y370H, Y372P, D373R, D373L,D373N, D373A, D373E, S374C, S374Q, S374T, S374A, Y375W, Y375A, F376R,F376H, F376Q, F376E, F376K, D377S, D377R, D377H, D377N, D377A, R379C,E380D, E380N, E380R, E380L, K382R, K382L, K382N, K382Q, K382E, K382D,M383L, K384S, K384L, K384R, K384T, K384Q, K384E, K384D, N385P, N385T,D386H, R388E, E389D, E389R, K392F, K392M, K392R, K392L, K392Y, K392H,K392E, K393F, K393L, K393R, K393Y, K393T, K393W, K393H, K393Q, K393A,K393E, K396F, K396M, K396L, K396R, K396Q, K396A, K396E, K396I, R397S,R397L, R397H, R397Q, R397E, R397K, R397I, E400Q, E400R, E400Y, E400L,S403F, S403R, S403L, S403Y, S403T, S403W, S403H, S403A, S403E, N407F,N407R, N407L, N407Y, N407W, N407H, N407Q, N407E, N407D, N407I, L408R,L408T, L408G, L408W, L408N, or L408D; or combinations thereof.

In other embodiments, the polypeptide comprises one or more ofmodifications at the amino acid residue corresponding to position C17,S18, E41, T43, P44, H45, Q46, Y53, D84, H85, L89, Q92, H119, D121, C122,S123, M124, T166, D167, V168, P169, I170, A171, G172, G173, G174, E175,E176, E177, G178, V210, M212, F217, E245, H247, S248, T249, D250, K269,V270, G271, P272, Y319, L335, D336, R337, R339, or Y340 of SEQ ID NO: 6;or combinations thereof.

In further embodiments, the polypeptide comprises one or more ofmodifications: (1) the amino acid residue corresponding to position C17of SEQ ID NO:6 is ALA, or THR; (2) the amino acid residue correspondingto position S18 of SEQ ID NO:6 is ALA, or ASN; (3) the amino acidresidue corresponding to position E41 of SEQ ID NO:6 is ALA, or GLN; (4)the amino acid residue corresponding to position H45 of SEQ ID NO:6 isALA, GLN, LYS, or PHE; (5) the amino acid residue corresponding toposition Q46 of SEQ ID NO:6 is ALA, ARG, SER, THR, or TRP; (6) the aminoacid residue corresponding to position Y53 of SEQ ID NO:6 is ALA, PHE,SER, or VAL; (7) the amino acid residue corresponding to position D84 ofSEQ ID NO:6 is ALA, ASN, GLY, or PRO; (8) the amino acid residuecorresponding to position H85 of SEQ ID NO:6 is ALA; (9) the amino acidresidue corresponding to position L89 of SEQ ID NO:6 is GLY, or SER;(10) the amino acid residue corresponding to position Q92 of SEQ ID NO:6is ALA, ASP, GLY, MET, or PRO; (11) the amino acid residue correspondingto position H119 of SEQ ID NO:6 is ALA, ASN, GLY, PRO, or SER; (12) theamino acid residue corresponding to position D121 of SEQ ID NO:6 is ALA,or ASN; (13) the amino acid residue corresponding to position C122 ofSEQ ID NO:6 is GLU, GLY, or LYS; (14) the amino acid residuecorresponding to position S123 of SEQ ID NO:6 is ALA, ARG, MET, or THR;(15) the amino acid residue corresponding to position M124 of SEQ IDNO:6 is ARG, or LYS; (16) the amino acid residue corresponding toposition T166 of SEQ ID NO:6 is ILE, LEU, or MET; (17) the amino acidresidue corresponding to position D167 of SEQ ID NO:6 is ALA, ASN, GLN,THR, or VAL; (18) the amino acid residue corresponding to position V168of SEQ ID NO:6 is ARG, or MET; (19) the amino acid residue correspondingto position I170 of SEQ ID NO:6 is LYS, MET, or THR; (20) the amino acidresidue corresponding to position A171 of SEQ ID NO:6 is PRO; (21) theamino acid residue corresponding to position G173 of SEQ ID NO:6 is SER;(22) the amino acid residue corresponding to position E175 of SEQ IDNO:6 is ARG, LYS, or VAL; (23) the amino acid residue corresponding toposition E176 of SEQ ID NO:6 is PHE, or TYR; (24) the amino acid residuecorresponding to position G178 of SEQ ID NO:6 is PRO; (25) the aminoacid residue corresponding to position V210 of SEQ ID NO:6 is PRO, orTHR; (26) the amino acid residue corresponding to position M212 of SEQID NO:6 is ARG, LEU, or LYS; (27) the amino acid residue correspondingto position E245 of SEQ ID NO:6 is ALA, or GLN; (28) the amino acidresidue corresponding to position H247 of SEQ ID NO:6 is ALA; (29) theamino acid residue corresponding to position T249 of SEQ ID NO:6 is SER;(30) the amino acid residue corresponding to position D250 of SEQ IDNO:6 is ALA, or ASN; (31) the amino acid residue corresponding toposition G271 of SEQ ID NO:6 is ALA, or ASP; (32) the amino acid residuecorresponding to position R339 of SEQ ID NO:6 is ALA, GLU, GLY, or THR;or (33) the amino acid residue corresponding to position Y340 of SEQ IDNO:6 is ALA, ARG, HIS, PHE, or PRO; or combinations thereof.

In some embodiments, the polypeptide comprises one or more ofmodifications: C17A, C17T, S18A, S18N, E41A, E41Q, H45F, H45A, H45K,H45Q, Q46R, Q46S, Q46A, Q46W, Q46T, Y53F, Y53A, Y53S, Y53V, D84G, D84A,D84N, D84P, H85A, L89G, L89S, Q92M, Q92D, Q92P, Q92G, Q92A, H119N,H119S, H119P, H119G, H119A, D121A, D121N, C122G, C122K, C122E, S123A,S123M, S123R, S123T, M124K, M124R, T166L, T166M, T166I, D167N, D167V,D167A, D167Q, D167T, V168M, V168R, I170M, I170K, I170T, A171P, G173S,E175K, E175V, E175R, E176F, E176Y, G178P, V210P, V210T, M212L, M212K,M212R, E245A, E245Q, H247A, T249S, D250A, D250N, G271D, G271A, R339G,R339A, R339E, R339T, Y340F, Y340H, Y340R, Y340P, or Y340A; orcombinations thereof.

In other embodiments, the polypeptide comprises one or more ofmodifications at the amino acid residue corresponding to position V13,Y14, S15, I16, S19, S21, Y22, S27, F30, K34, G35, D36, Y37, S54, K62,N63, M66, K67, K72, E75, E76, D77, K78, D93, S96, P97, T98, N101, D105,F110, S113, Y115, L126, S127, D128, D129, K131, Y135, E136, E140, T142,R143, E144, F146, E147, I148, E151, T152, R154, P160, V179, V182, D184,S187, A188, I189, S190, S191, L192, Y195, N197, D198, V199, P200, N201,I202, D204, G208, I211, L213, I215, S218, Y219, V230, G232, I233, L234,V237, E240, D241, L242, V244, G246, A253, Y255, A256, N259, E262, R266,A273, R280, G281, V282, L284, S286, N287, D290, E291, I292, E295, R296,E297, K302, R303, K321, K324, R325, E327, L328, I330, W331, N333, L334,E343, Y344, E345, M349, V350, N352, K353, F358, E360, V362, D363, I364,K365, F366, Y372, D373, S374, F376, D377, R379, M383, N385, K393, R397,or S403 of SEQ ID NO:6; or combinations thereof.

In further embodiments, the polypeptide comprises one or more ofmodifications: (1) the amino acid residue corresponding to position V13of SEQ ID NO:6 is ILE; (2) the amino acid residue corresponding toposition Y14 of SEQ ID NO:6 is PHE; (3) the amino acid residuecorresponding to position S15 of SEQ ID NO:6 is ALA; (4) the amino acidresidue corresponding to position I16 of SEQ ID NO:6 is VAL; (5) theamino acid residue corresponding to position S19 of SEQ ID NO:6 is ASN;(6) the amino acid residue corresponding to position S21 of SEQ ID NO:6is GLU, LYS, or PRO; (7) the amino acid residue corresponding toposition Y22 of SEQ ID NO:6 is TRP; (8) the amino acid residuecorresponding to position S27 of SEQ ID NO:6 is ALA, or CYS; (9) theamino acid residue corresponding to position F30 of SEQ ID NO:6 is HIS;(10) the amino acid residue corresponding to position K34 of SEQ ID NO:6is ARG, ASP, MET, or TYR; (11) the amino acid residue corresponding toposition G35 of SEQ ID NO:6 is ASP, or HIS; (12) the amino acid residuecorresponding to position D36 of SEQ ID NO:6 is SER, or THR; (13) theamino acid residue corresponding to position Y37 of SEQ ID NO:6 is PRO;(14) the amino acid residue corresponding to position S54 of SEQ ID NO:6is THR; (15) the amino acid residue corresponding to position K62 of SEQID NO:6 is ARG; (16) the amino acid residue corresponding to positionN63 of SEQ ID NO:6 is ARG; (17) the amino acid residue corresponding toposition M66 of SEQ ID NO:6 is PHE, or TYR; (18) the amino acid residuecorresponding to position K67 of SEQ ID NO:6 is GLN; (19) the amino acidresidue corresponding to position K72 of SEQ ID NO:6 is LEU, or VAL;(20) the amino acid residue corresponding to position E75 of SEQ ID NO:6is PRO; (21) the amino acid residue corresponding to position E76 of SEQID NO:6 is PRO; (22) the amino acid residue corresponding to positionD77 of SEQ ID NO:6 is ASN; (23) the amino acid residue corresponding toposition K78 of SEQ ID NO:6 is ARG; (24) the amino acid residuecorresponding to position D93 of SEQ ID NO:6 is HIS; (25) the amino acidresidue corresponding to position S96 of SEQ ID NO:6 is ALA, or GLU;(26) the amino acid residue corresponding to position P97 of SEQ ID NO:6is ASP, or GLU; (27) the amino acid residue corresponding to positionT98 of SEQ ID NO:6 is GLN, or GLU; (28) the amino acid residuecorresponding to position N101 of SEQ ID NO:6 is ALA, GLU, or LYS; (29)the amino acid residue corresponding to position D105 of SEQ ID NO:6 isGLU; (30) the amino acid residue corresponding to position F110 of SEQID NO:6 is TYR; (31) the amino acid residue corresponding to positionS113 of SEQ ID NO:6 is ALA; (32) the amino acid residue corresponding toposition Y115 of SEQ ID NO:6 is PHE; (33) the amino acid residuecorresponding to position L126 of SEQ ID NO:6 is CYS; (34) the aminoacid residue corresponding to position S127 of SEQ ID NO:6 is ALA; (35)the amino acid residue corresponding to position D128 of SEQ ID NO:6 isGLY; (36) the amino acid residue corresponding to position D129 of SEQID NO:6 is GLU; (37) the amino acid residue corresponding to positionK131 of SEQ ID NO:6 is ALA; (38) the amino acid residue corresponding toposition Y135 of SEQ ID NO:6 is ASN, ASP, GLN, LEU, LYS, MET, PRO, orTHR; (39) the amino acid residue corresponding to position E136 of SEQID NO:6 is ASP; (40) the amino acid residue corresponding to positionE140 of SEQ ID NO:6 is ILE; (41) the amino acid residue corresponding toposition T142 of SEQ ID NO:6 is GLU; (42) the amino acid residuecorresponding to position R143 of SEQ ID NO:6 is ALA, or VAL; (43) theamino acid residue corresponding to position E144 of SEQ ID NO:6 is ARG;(44) the amino acid residue corresponding to position F146 of SEQ IDNO:6 is CYS; (45) the amino acid residue corresponding to position E147of SEQ ID NO:6 is ARG, or LYS; (46) the amino acid residue correspondingto position I148 of SEQ ID NO:6 is VAL; (47) the amino acid residuecorresponding to position E151 of SEQ ID NO:6 is ARG; (48) the aminoacid residue corresponding to position T152 of SEQ ID NO:6 is ALA; (49)the amino acid residue corresponding to position R154 of SEQ ID NO:6 isGLN; (50) the amino acid residue corresponding to position P160 of SEQID NO:6 is ALA, or GLU; (51) the amino acid residue corresponding toposition V179 of SEQ ID NO:6 is ILE; (52) the amino acid residuecorresponding to position V182 of SEQ ID NO:6 is ALA, LYS, or PRO; (53)the amino acid residue corresponding to position D184 of SEQ ID NO:6 isGLU; (54) the amino acid residue corresponding to position S187 of SEQID NO:6 is ARG, ASN, GLU, LYS, or THR; (55) the amino acid residuecorresponding to position A188 of SEQ ID NO:6 is MET, PHE, or THR; (56)the amino acid residue corresponding to position I189 of SEQ ID NO:6 isLEU; (57) the amino acid residue corresponding to position S190 of SEQID NO:6 is ASP, or GLU; (58) the amino acid residue corresponding toposition S191 of SEQ ID NO:6 is ALA, ILE, LEU, THR, TYR, or VAL; (59)the amino acid residue corresponding to position L192 of SEQ ID NO:6 isHIS, or PHE; (60) the amino acid residue corresponding to position Y195of SEQ ID NO:6 is ALA; (61) the amino acid residue corresponding toposition N197 of SEQ ID NO:6 is ALA, ASP, GLN, GLU, or LYS; (62) theamino acid residue corresponding to position D198 of SEQ ID NO:6 is LYS;(63) the amino acid residue corresponding to position V199 of SEQ IDNO:6 is CYS, HIS, ILE, or LEU; (64) the amino acid residue correspondingto position P200 of SEQ ID NO:6 is GLY; (65) the amino acid residuecorresponding to position N201 of SEQ ID NO:6 is ARG, GLN, or LYS; (66)the amino acid residue corresponding to position I202 of SEQ ID NO:6 isALA, or MET; (67) the amino acid residue corresponding to position D204of SEQ ID NO:6 is ASN; (68) the amino acid residue corresponding toposition G208 of SEQ ID NO:6 is ALA; (69) the amino acid residuecorresponding to position I211 of SEQ ID NO:6 is VAL; (70) the aminoacid residue corresponding to position L213 of SEQ ID NO:6 is MET; (71)the amino acid residue corresponding to position I215 of SEQ ID NO:6 isVAL; (72) the amino acid residue corresponding to position S218 of SEQID NO:6 is GLY; (73) the amino acid residue corresponding to positionY219 of SEQ ID NO:6 is ASP, or HIS; (74) the amino acid residuecorresponding to position V230 of SEQ ID NO:6 is ALA; (75) the aminoacid residue corresponding to position G232 of SEQ ID NO:6 is ASP, GLU,LYS, or PRO; (76) the amino acid residue corresponding to position I233of SEQ ID NO:6 is LEU; (77) the amino acid residue corresponding toposition L234 of SEQ ID NO:6 is ALA, or MET; (78) the amino acid residuecorresponding to position V237 of SEQ ID NO:6 is LEU; (79) the aminoacid residue corresponding to position E240 of SEQ ID NO:6 is THR; (80)the amino acid residue corresponding to position D241 of SEQ ID NO:6 isGLY, or PRO; (81) the amino acid residue corresponding to position L242of SEQ ID NO:6 is TRP; (82) the amino acid residue corresponding toposition V244 of SEQ ID NO:6 is LEU; (83) the amino acid residuecorresponding to position G246 of SEQ ID NO:6 is ALA, CYS, or VAL; (84)the amino acid residue corresponding to position A253 of SEQ ID NO:6 isASN, ASP, PRO, SER, or THR; (85) the amino acid residue corresponding toposition Y255 of SEQ ID NO:6 is ASP, GLN, GLU, or LYS; (86) the aminoacid residue corresponding to position A256 of SEQ ID NO:6 is LEU; (87)the amino acid residue corresponding to position N259 of SEQ ID NO:6 isALA, ARG, ASP, GLU, or TRP; (88) the amino acid residue corresponding toposition E262 of SEQ ID NO:6 is GLN; (89) the amino acid residuecorresponding to position R266 of SEQ ID NO:6 is ALA, or VAL; (90) theamino acid residue corresponding to position A273 of SEQ ID NO:6 is TRP;(91) the amino acid residue corresponding to position R280 of SEQ IDNO:6 is GLU; (92) the amino acid residue corresponding to position G281of SEQ ID NO:6 is ALA; (93) the amino acid residue corresponding toposition V282 of SEQ ID NO:6 is ILE; (94) the amino acid residuecorresponding to position L284 of SEQ ID NO:6 is LYS; (95) the aminoacid residue corresponding to position S286 of SEQ ID NO:6 is ALA, ASP,or VAL; (96) the amino acid residue corresponding to position N287 ofSEQ ID NO:6 is ASP; (97) the amino acid residue corresponding toposition D290 of SEQ ID NO:6 is ALA, ASN, GLN, or LYS; (98) the aminoacid residue corresponding to position E291 of SEQ ID NO:6 is ASP; (99)the amino acid residue corresponding to position I292 of SEQ ID NO:6 isLEU; (100) the amino acid residue corresponding to position E295 of SEQID NO:6 is PRO; (101) the amino acid residue corresponding to positionR296 of SEQ ID NO:6 is ASP, GLU, or TYR; (102) the amino acid residuecorresponding to position E297 of SEQ ID NO:6 is ARG, GLN, GLY, HIS, orLYS; (103) the amino acid residue corresponding to position K302 of SEQID NO:6 is LEU; (104) the amino acid residue corresponding to positionR303 of SEQ ID NO:6 is GLU; (105) the amino acid residue correspondingto position K321 of SEQ ID NO:6 is ARG, HIS, or THR; (106) the aminoacid residue corresponding to position K324 of SEQ ID NO:6 is ASP, orGLU; (107) the amino acid residue corresponding to position R325 of SEQID NO:6 is ASN, ASP, GLN, or GLU; (108) the amino acid residuecorresponding to position E327 of SEQ ID NO:6 is ARG; (109) the aminoacid residue corresponding to position L328 of SEQ ID NO:6 is PHE, orTRP; (110) the amino acid residue corresponding to position I330 of SEQID NO:6 is LEU; (111) the amino acid residue corresponding to positionW331 of SEQ ID NO:6 is HIS; (112) the amino acid residue correspondingto position N333 of SEQ ID NO:6 is ALA, or SER; (113) the amino acidresidue corresponding to position L334 of SEQ ID NO:6 is TYR; (114) theamino acid residue corresponding to position E343 of SEQ ID NO:6 is ASN;(115) the amino acid residue corresponding to position Y344 of SEQ IDNO:6 is HIS; (116) the amino acid residue corresponding to position E345of SEQ ID NO:6 is PRO; (117) the amino acid residue corresponding toposition M349 of SEQ ID NO:6 is ALA, ARG, CYS, GLU, LYS, or THR; (118)the amino acid residue corresponding to position V350 of SEQ ID NO:6 isALA; (119) the amino acid residue corresponding to position N352 of SEQID NO:6 is GLU; (120) the amino acid residue corresponding to positionK353 of SEQ ID NO:6 is ARG; (121) the amino acid residue correspondingto position F358 of SEQ ID NO:6 is LEU; (122) the amino acid residuecorresponding to position E360 of SEQ ID NO:6 is ASP; (123) the aminoacid residue corresponding to position V362 of SEQ ID NO:6 is ILE; (124)the amino acid residue corresponding to position D363 of SEQ ID NO:6 isPRO; (125) the amino acid residue corresponding to position I364 of SEQID NO:6 is TRP; (126) the amino acid residue corresponding to positionK365 of SEQ ID NO:6 is ARG, or THR; (127) the amino acid residuecorresponding to position F366 of SEQ ID NO:6 is LEU; (128) the aminoacid residue corresponding to position Y372 of SEQ ID NO:6 is PRO; (129)the amino acid residue corresponding to position D373 of SEQ ID NO:6 isALA; (130) the amino acid residue corresponding to position S374 of SEQID NO:6 is ALA, CYS, GLN, or THR; (131) the amino acid residuecorresponding to position F376 of SEQ ID NO:6 is ARG, GLN, GLU, HIS, orLYS; (132) the amino acid residue corresponding to position D377 of SEQID NO:6 is ALA; (133) the amino acid residue corresponding to positionR379 of SEQ ID NO:6 is CYS; (134) the amino acid residue correspondingto position M383 of SEQ ID NO:6 is LEU; (135) the amino acid residuecorresponding to position N385 of SEQ ID NO:6 is PRO; (136) the aminoacid residue corresponding to position K393 of SEQ ID NO:6 is HIS; (137)the amino acid residue corresponding to position R397 of SEQ ID NO:6 isLYS; or (138) the amino acid residue corresponding to position S403 ofSEQ ID NO:6 is ALA, HIS, or TYR; or combinations thereof.

In some embodiments, the polypeptide comprises one or more ofmodifications: V13I, Y14F, S15A, I16V, S19N, S21P, S21E, S21K, Y22W,S27A, S27C, F30H, K34D, K34M, K34R, K34Y, G35D, G35H, D36T, D36S, Y37P,S54T, K62R, N63R, M66F, M66Y, K67Q, K72V, K72L, E75P, E76P, D77N, K78R,D93H, S96E, S96A, P97D, P97E, T98E, T98Q, N101E, N101A, N101K, D105E,F110Y, S113A, Y115F, L126C, S127A, D128G, D129E, K131A, Y135P, Y135Q,Y135K, Y135N, Y135T, Y135M, Y135D, Y135L, E136D, E140I, T142E, R143V,R143A, E144R, F146C, E147R, E147K, I148V, E151R, T152A, R154Q, P160E,P160A, V179I, V182P, V182A, V182K, D184E, S187K, S187N, S187T, S187E,S187R, A188F, A188M, A188T, I189L, S190D, S190E, S191I, S191T, S191V,S191Y, S191A, S191L, L192F, L192H, Y195A, N197Q, N197K, N197E, N197D,N197A, D198K, V199I, V199H, V199L, V199C, P200G, N201R, N201Q, N201K,I202M, I202A, D204N, G208A, I211V, L213M, I215V, S218G, Y219D, Y219H,V230A, G232D, G232E, G232K, G232P, I233L, L234M, L234A, V237L, E240T,D241P, D241G, L242W, V244L, G246V, G246A, G246C, A253P, A253N, A253T,A253D, A253S, Y255D, Y255E, Y255Q, Y255K, A256L, N259R, N259E, N259W,N259D, N259A, E262Q, R266V, R266A, A273W, R280E, G281A, V282I, L284K,S286V, S286D, S286A, N287D, D290N, D290A, D290Q, D290K, E291D, I292L,E295P, R296D, R296E, R296Y, E297Q, E297K, E297G, E297H, E297R, K302L,R303E, K321T, K321H, K321R, K324D, K324E, R325D, R325N, R325E, R325Q,E327R, L328F, L328W, I330L, W331H, N333A, N333S, L334Y, E343N, Y344H,E345P, M349K, M349C, M349T, M349E, M349R, M349A, V350A, N352E, K353R,F358L, E360D, V362I, D363P, I364W, K365T, K365R, F366L, Y372P, D373A,S374T, S374Q, S374A, S374C, F376Q, F376K, F376E, F376H, F376R, D377A,R379C, M383L, N385P, K393H, R397K, S403H, S403Y, or S403A; orcombinations thereof.

In other embodiments, the polypeptide comprises one or more ofmodifications at the amino acid residue corresponding to position K5,K10, K11, C17, K32, K34, K62, K67, K70, K72, K78, K102, K104, K116,K117, C122, K131, K137, K155, K193, K194, K221, K229, K238, K269, K302,K318, K321, K324, K348, K353, K365, K382, K384, K392, K393, or K396 ofSEQ ID NO:6; or combinations thereof.

In further embodiments, the polypeptide comprises one or more ofmodifications: (1) the amino acid residue corresponding to position K5of SEQ ID NO:6 is ALA, ARG, HIS, LEU, MET, PHE, or TRP; (2) the aminoacid residue corresponding to position K10 of SEQ ID NO:6 is ALA, ARG,ASN, LEU, MET, or VAL; (3) the amino acid residue corresponding toposition K11 of SEQ ID NO:6 is ARG, GLN, GLU, LEU, TYR, or VAL; (4) theamino acid residue corresponding to position C17 of SEQ ID NO:6 is ALA,ARG, GLY, ILE, LEU, SER, THR, or VAL; (5) the amino acid residuecorresponding to position K32 of SEQ ID NO:6 is ARG, GLN, ILE, LEU, MET,or SER; (6) the amino acid residue corresponding to position K34 of SEQID NO:6 is ARG, GLN, GLU, HIS, LEU, MET, PHE, THR, or TYR; (7) the aminoacid residue corresponding to position K62 of SEQ ID NO:6 is ARG, TYR,or VAL; (8) the amino acid residue corresponding to position K67 of SEQID NO:6 is ARG, GLN, SER, or THR; (9) the amino acid residuecorresponding to position K70 of SEQ ID NO:6 is ARG, or GLN; (10) theamino acid residue corresponding to position K72 of SEQ ID NO:6 is ARG,ILE, LEU, THR, or VAL; (11) the amino acid residue corresponding toposition K78 of SEQ ID NO:6 is ARG, GLN, LEU, or MET; (12) the aminoacid residue corresponding to position K102 of SEQ ID NO:6 is ARG, LEU,or TYR; (13) the amino acid residue corresponding to position K104 ofSEQ ID NO:6 is ARG, ASN, GLN, ILE, PHE, or VAL; (14) the amino acidresidue corresponding to position K116 of SEQ ID NO:6 is ARG, GLN, HIS,ILE, LEU, SER, THR, TRP, or TYR; (15) the amino acid residuecorresponding to position K117 of SEQ ID NO:6 is ARG, LEU, MET, TRP, orVAL; (16) the amino acid residue corresponding to position C122 of SEQID NO:6 is ALA, ARG, THR, or VAL; (17) the amino acid residuecorresponding to position K131 of SEQ ID NO:6 is ARG, GLN, GLU, LEU, orTYR; (18) the amino acid residue corresponding to position K137 of SEQID NO:6 is ARG, GLN, GLU, ILE, LEU, or THR; (19) the amino acid residuecorresponding to position K155 of SEQ ID NO:6 is ARG, ASN, ASP, GLU,HIS, or TYR; (20) the amino acid residue corresponding to position K193of SEQ ID NO:6 is ALA, ARG, GLN, GLU, or ILE; (21) the amino acidresidue corresponding to position K194 of SEQ ID NO:6 is ALA, ARG, GLU,SER, or THR; (22) the amino acid residue corresponding to position K221of SEQ ID NO:6 is ARG, GLN, ILE, THR, or VAL; (23) the amino acidresidue corresponding to position K229 of SEQ ID NO:6 is ALA, ARG, ASN,GLN, SER, or THR; (24) the amino acid residue corresponding to positionK238 of SEQ ID NO:6 is ARG, SER, or TRP; (25) the amino acid residuecorresponding to position K269 of SEQ ID NO:6 is ARG, ASN, ILE, LEU, orVAL; (26) the amino acid residue corresponding to position K302 of SEQID NO:6 is ARG, GLN, ILE, LEU, or MET; (27) the amino acid residuecorresponding to position K318 of SEQ ID NO:6 is ARG, ASP, or GLU; (28)the amino acid residue corresponding to position K321 of SEQ ID NO:6 isARG, or ASP; (29) the amino acid residue corresponding to position K324of SEQ ID NO:6 is ARG, ASP, GLU, or SER; (30) the amino acid residuecorresponding to position K348 of SEQ ID NO:6 is ARG, LEU, or VAL; (31)the amino acid residue corresponding to position K353 of SEQ ID NO:6 isARG, GLN, LEU, or TRP; (32) the amino acid residue corresponding toposition K365 of SEQ ID NO:6 is ARG, HIS, or SER; (33) the amino acidresidue corresponding to position K382 of SEQ ID NO:6 is ARG, ASN, orLEU; (34) the amino acid residue corresponding to position K384 of SEQID NO:6 is ARG, ASP, GLN, GLU, LEU, SER, or THR; (35) the amino acidresidue corresponding to position K392 of SEQ ID NO:6 is ARG, LEU, orMET; (36) the amino acid residue corresponding to position K393 of SEQID NO:6 is ARG, HIS, LEU, PHE, or TYR; or (37) the amino acid residuecorresponding to position K396 of SEQ ID NO:6 is ARG, ILE, LEU, MET, orPHE; or combinations thereof.

In some embodiments, the polypeptide comprises one or more ofmodifications: K5H, K5M, K5L, K5A, K5R, K5W, K5F, K10M, K10L, K10R,K10A, K10V, K10N, K11Q, K11Y, K11E, K11L, K11R, K11V, C17T, C17I, C17L,C17A, C17R, C17V, C17G, C17S, K32Q, K32I, K32M, K32L, K32R, K32S, K34T,K34Q, K34Y, K34E, K34H, K34M, K34L, K34R, K34F, K62R, K62Y, K62V, K67R,K67T, K67Q, K67S, K70R, K70Q, K72T, K72I, K72L, K72R, K72V, K78R, K78Q,K78M, K78L, K102R, K102Y, K102L, K104Q, K104I, K104R, K104V, K104N,K104F, K116T, K116Q, K116Y, K116H, K116I, K116L, K116R, K116W, K116S,K117M, K117L, K117R, K117V, K117W, C122R, C122T, C122A, C122V, K131Q,K131Y, K131E, K131L, K131R, K137T, K137Q, K137E, K137I, K137L, K137R,K155Y, K155E, K155H, K155R, K155D, K155N, K193Q, K193E, K193I, K193R,K193A, K194T, K194E, K194R, K194A, K194S, K221Q, K221T, K221I, K221R,K221V, K229T, K229Q, K229R, K229A, K229N, K229S, K238R, K238W, K238S,K269I, K269L, K269R, K269V, K269N, K302Q, K302I, K302M, K302L, K302R,K318R, K318D, K318E, K321R, K321D, K324R, K324D, K324E, K324S, K348V,K348R, K348L, K353W, K353R, K353Q, K353L, K365R, K365H, K365S, K382R,K382N, K382L, K384T, K384Q, K384E, K384L, K384D, K384R, K384S, K392R,K392M, K392L, K393Y, K393H, K393L, K393R, K393F, K396I, K396M, K396L,K396R, or K396F; or combinations thereof.

In other embodiments, the polypeptide comprises one or more ofmodifications at the amino acid residue corresponding to position L3,S4, K5, D6, Y7, L8, R9, K10, K11, S21, Y22, E25, E29, F30, K32, E33,K34, G35, D36, Y37, Q49, F50, M56, E59, K62, N63, F64, M66, K67, K70,E71, K72, E75, E76, D77, K78, I80, E94, S96, T98, N101, K102, K104,D105, R108, K116, P125, D129, P130, K131, V132, P134, Y135, E136, K137,E140, R141, E144, E147, I148, E150, E151, T152, A153, R154, K155, Y156,N157, F158, Q159, P160, V161, V179, S187, S190, S191, K193, K194, Y195,N197, P200, N201, W203, D204, R205, G216, S218, Y219, D220, K221, V222,E224, D228, K229, R231, G232, E235, K238, R239, E240, D241, R258, E262,N287, D290, E291, I292, E297, S299, N300, K302, R303, E307, D313, Y315,K318, K321, D322, S323, K324, R325, L326, E327, L328, W331, E343, E345,K348, M349, N352, K353, E356, N357, G361, D363, I364, K365, F366, Y368,Q369, Y370, D373, Y375, F376, D377, E380, K382, K384, N385, D386, R388,E389, K392, K393, K396, R397, E400, S403, N407, or L408 of SEQ ID NO:6;or combinations thereof.

In further embodiments, the polypeptide comprises one or more ofmodifications: (1) the amino acid residue corresponding to position L3of SEQ ID NO:6 is GLU, or THR; (2) the amino acid residue correspondingto position S4 of SEQ ID NO:6 is ARG, ASN, ASP, GLN, GLU, LEU, MET, orPRO; (3) the amino acid residue corresponding to position K5 of SEQ IDNO:6 is ALA, ARG, GLN, HIS, LEU, PHE, or VAL; (4) the amino acid residuecorresponding to position D6 of SEQ ID NO:6 is ALA, ARG, ASN, GLN, GLU,HIS, or LEU; (5) the amino acid residue corresponding to position Y7 ofSEQ ID NO:6 is ASN, HIS, or TRP; (6) the amino acid residuecorresponding to position L8 of SEQ ID NO:6 is ASN, or GLY; (7) theamino acid residue corresponding to position R9 of SEQ ID NO:6 is GLN,GLU, HIS, ILE, LEU, or THR; (8) the amino acid residue corresponding toposition K10 of SEQ ID NO:6 is ALA, ARG, ASN, ASP, GLU, or PRO; (9) theamino acid residue corresponding to position K11 of SEQ ID NO:6 is ARG,GLN, GLU, HIS, ILE, PHE, TRP, TYR, or VAL; (10) the amino acid residuecorresponding to position S21 of SEQ ID NO:6 is ARG, GLN, GLU, or PRO;(11) the amino acid residue corresponding to position Y22 of SEQ ID NO:6is TRP; (12) the amino acid residue corresponding to position E25 of SEQID NO:6 is ARG, or ILE; (13) the amino acid residue corresponding toposition E29 of SEQ ID NO:6 is ARG, GLN, ILE, or LEU; (14) the aminoacid residue corresponding to position F30 of SEQ ID NO:6 is ALA, HIS,or THR; (15) the amino acid residue corresponding to position K32 of SEQID NO:6 is ALA, ARG, GLN, ILE, LEU, or MET; (16) the amino acid residuecorresponding to position E33 of SEQ ID NO:6 is ARG, ASN, ASP, GLN, HIS,or THR; (17) the amino acid residue corresponding to position K34 of SEQID NO:6 is ARG, ASP, GLN, HIS, ILE, LEU, MET, PHE, SER, THR, TYR, orVAL; (18) the amino acid residue corresponding to position G35 of SEQ IDNO:6 is ASN, ASP, HIS, or SER; (19) the amino acid residue correspondingto position D36 of SEQ ID NO:6 is ARG, ASN, GLN, GLU, HIS, LEU, PHE,SER, THR, TRP, or TYR; (20) the amino acid residue corresponding toposition Y37 of SEQ ID NO:6 is ARG, ASP, GLU, or PRO; (21) the aminoacid residue corresponding to position Q49 of SEQ ID NO:6 is HIS, ILE,PHE, or TRP; (22) the amino acid residue corresponding to position F50of SEQ ID NO:6 is ARG, GLN, HIS, or SER; (23) the amino acid residuecorresponding to position M56 of SEQ ID NO:6 is TRP; (24) the amino acidresidue corresponding to position E59 of SEQ ID NO:6 is ARG, SER, orTHR; (25) the amino acid residue corresponding to position K62 of SEQ IDNO:6 is ALA, or ARG; (26) the amino acid residue corresponding toposition N63 of SEQ ID NO:6 is ARG, ASP, GLN, or GLU; (27) the aminoacid residue corresponding to position F64 of SEQ ID NO:6 is GLU, orLEU; (28) the amino acid residue corresponding to position M66 of SEQ IDNO:6 is GLU, PHE, or TYR; (29) the amino acid residue corresponding toposition K67 of SEQ ID NO:6 is ARG, ASP, GLN, GLU, SER, or THR; (30) theamino acid residue corresponding to position K70 of SEQ ID NO:6 is ARG,ASP, GLN, or LEU; (31) the amino acid residue corresponding to positionE71 of SEQ ID NO:6 is ARG, GLN, or SER; (32) the amino acid residuecorresponding to position K72 of SEQ ID NO:6 is GLN, GLU, HIS, ILE, LEU,or THR; (33) the amino acid residue corresponding to position E75 of SEQID NO:6 is ARG, ASP, or SER; (34) the amino acid residue correspondingto position E76 of SEQ ID NO:6 is ARG, LEU, or PRO; (35) the amino acidresidue corresponding to position D77 of SEQ ID NO:6 is ARG, ASN, orSER; (36) the amino acid residue corresponding to position K78 of SEQ IDNO:6 is ARG, ASP, GLN, LEU, or MET; (37) the amino acid residuecorresponding to position I80 of SEQ ID NO:6 is ARG, GLN, or LEU; (38)the amino acid residue corresponding to position E94 of SEQ ID NO:6 isARG, GLN, LEU, or TYR; (39) the amino acid residue corresponding toposition S96 of SEQ ID NO:6 is ALA, ARG, or GLU; (40) the amino acidresidue corresponding to position T98 of SEQ ID NO:6 is ARG, GLN, GLU,ILE, TRP, or VAL; (41) the amino acid residue corresponding to positionN101 of SEQ ID NO:6 is ALA, ARG, ASP, GLN, GLU, HIS, SER, or THR; (42)the amino acid residue corresponding to position K102 of SEQ ID NO:6 isARG, GLN, GLU, TRP, or TYR; (43) the amino acid residue corresponding toposition K104 of SEQ ID NO:6 is ARG, GLN, or GLU; (44) the amino acidresidue corresponding to position D105 of SEQ ID NO:6 is GLN, GLU, orILE; (45) the amino acid residue corresponding to position R108 of SEQID NO:6 is ILE; (46) the amino acid residue corresponding to positionK116 of SEQ ID NO:6 is ARG, ASP, GLN, GLU, HIS, ILE, LEU, THR, TRP, orTYR; (47) the amino acid residue corresponding to position P125 of SEQID NO:6 is ALA; (48) the amino acid residue corresponding to positionD129 of SEQ ID NO:6 is ARG, GLN, GLU, LEU, or TRP; (49) the amino acidresidue corresponding to position P130 of SEQ ID NO:6 is SER; (50) theamino acid residue corresponding to position K131 of SEQ ID NO:6 is ASP,GLU, SER, or TYR; (51) the amino acid residue corresponding to positionV132 of SEQ ID NO:6 is ARG, GLU, SER, THR, or TYR; (52) the amino acidresidue corresponding to position P134 of SEQ ID NO:6 is ALA, ASP, orHIS; (53) the amino acid residue corresponding to position Y135 of SEQID NO:6 is ARG, ASP, GLN, LEU, or TRP; (54) the amino acid residuecorresponding to position E136 of SEQ ID NO:6 is ARG, ASP, GLN, PRO,SER, THR, or TRP; (55) the amino acid residue corresponding to positionK137 of SEQ ID NO:6 is ARG, GLN, GLU, ILE, LEU, TRP, or VAL; (56) theamino acid residue corresponding to position E140 of SEQ ID NO:6 is ARG,ASP, ILE, or LEU; (57) the amino acid residue corresponding to positionR141 of SEQ ID NO:6 is ASP, GLN, HIS, LEU, TRP, or TYR; (58) the aminoacid residue corresponding to position E144 of SEQ ID NO:6 is ARG, ASP,GLN, or ILE; (59) the amino acid residue corresponding to position E147of SEQ ID NO:6 is ALA, ARG, or GLN; (60) the amino acid residuecorresponding to position I148 of SEQ ID NO:6 is ASP, GLU, or VAL; (61)the amino acid residue corresponding to position E150 of SEQ ID NO:6 isALA, or ARG; (62) the amino acid residue corresponding to position E151of SEQ ID NO:6 is ARG; (63) the amino acid residue corresponding toposition T152 of SEQ ID NO:6 is ALA, ARG, GLN, HIS, TYR, or VAL; (64)the amino acid residue corresponding to position A153 of SEQ ID NO:6 isARG, GLN, GLY, or SER; (65) the amino acid residue corresponding toposition R154 of SEQ ID NO:6 is GLN, GLU, PRO, SER, or THR; (66) theamino acid residue corresponding to position K155 of SEQ ID NO:6 is ASP,GLN, GLU, GLY, PRO, THR, or TRP; (67) the amino acid residuecorresponding to position Y156 of SEQ ID NO:6 is ARG, ASP, GLN, GLU,MET, PHE, THR, or VAL; (68) the amino acid residue corresponding toposition N157 of SEQ ID NO:6 is ARG, ASP, GLU, or PRO; (69) the aminoacid residue corresponding to position F158 of SEQ ID NO:6 is ARG, ASN,GLU, LEU, THR, or VAL; (70) the amino acid residue corresponding toposition Q159 of SEQ ID NO:6 is ARG, GLU, GLY, LEU, THR, or TRP; (71)the amino acid residue corresponding to position P160 of SEQ ID NO:6 isALA, ARG, GLU, ILE, LEU, MET, PHE, SER, or THR; (72) the amino acidresidue corresponding to position V161 of SEQ ID NO:6 is GLU, ILE, TRP,or TYR; (73) the amino acid residue corresponding to position V179 ofSEQ ID NO:6 is ARG, or ILE; (74) the amino acid residue corresponding toposition S187 of SEQ ID NO:6 is ALA, ARG, or GLN; (75) the amino acidresidue corresponding to position S190 of SEQ ID NO:6 is ASP, GLU, orTYR; (76) the amino acid residue corresponding to position S191 of SEQID NO:6 is ALA, ARG, GLN, GLU, HIS, LEU, THR, TRP, or TYR; (77) theamino acid residue corresponding to position K193 of SEQ ID NO:6 is ARG,GLN, GLU, ILE, LEU, PHE, or TYR; (78) the amino acid residuecorresponding to position K194 of SEQ ID NO:6 is ALA, ARG, ASP, GLN,GLU, or LEU; (79) the amino acid residue corresponding to position Y195of SEQ ID NO:6 is ALA, GLN, or HIS; (80) the amino acid residuecorresponding to position N197 of SEQ ID NO:6 is ARG, ASP, GLN, GLU,LEU, PRO, or SER; (81) the amino acid residue corresponding to positionP200 of SEQ ID NO:6 is ARG, ASP, GLU, LEU, or MET; (82) the amino acidresidue corresponding to position N201 of SEQ ID NO:6 is ARG, or GLU;(83) the amino acid residue corresponding to position W203 of SEQ IDNO:6 is ALA, ARG, HIS, LEU, PHE, or TYR; (84) the amino acid residuecorresponding to position D204 of SEQ ID NO:6 is ARG, ASN, or SER; (85)the amino acid residue corresponding to position R205 of SEQ ID NO:6 isGLN, or LEU; (86) the amino acid residue corresponding to position G216of SEQ ID NO:6 is ARG, ASN, or THR; (87) the amino acid residuecorresponding to position S218 of SEQ ID NO:6 is THR; (88) the aminoacid residue corresponding to position Y219 of SEQ ID NO:6 is ARG, orGLY; (89) the amino acid residue corresponding to position D220 of SEQID NO:6 is ARG, GLN, GLU, or THR; (90) the amino acid residuecorresponding to position K221 of SEQ ID NO:6 is ARG, GLN, GLU, ILE,THR, or VAL; (91) the amino acid residue corresponding to position V222of SEQ ID NO:6 is ILE; (92) the amino acid residue corresponding toposition E224 of SEQ ID NO:6 is GLN, MET, or TYR; (93) the amino acidresidue corresponding to position D228 of SEQ ID NO:6 is GLU, or SER;(94) the amino acid residue corresponding to position K229 of SEQ IDNO:6 is ALA, ARG, ASN, GLN, or GLU; (95) the amino acid residuecorresponding to position R231 of SEQ ID NO:6 is ALA, GLU, LEU, or PHE;(96) the amino acid residue corresponding to position G232 of SEQ IDNO:6 is ARG, GLU, or PRO; (97) the amino acid residue corresponding toposition E235 of SEQ ID NO:6 is ARG, or TRP; (98) the amino acid residuecorresponding to position K238 of SEQ ID NO:6 is ARG, GLN, GLU, SER, orTRP; (99) the amino acid residue corresponding to position R239 of SEQID NO:6 is SER; (100) the amino acid residue corresponding to positionE240 of SEQ ID NO:6 is ASN, ASP, HIS, MET, THR, TYR, or VAL; (101) theamino acid residue corresponding to position D241 of SEQ ID NO:6 is ARG,ASN, GLN, or PRO; (102) the amino acid residue corresponding to positionR258 of SEQ ID NO:6 is GLU, or LEU; (103) the amino acid residuecorresponding to position E262 of SEQ ID NO:6 is ARG, GLN, or LEU; (104)the amino acid residue corresponding to position N287 of SEQ ID NO:6 isARG, GLN, GLU, HIS, LEU, or PHE; (105) the amino acid residuecorresponding to position D290 of SEQ ID NO:6 is ARG, GLN, GLU, or PHE;(106) the amino acid residue corresponding to position E291 of SEQ IDNO:6 is ARG; (107) the amino acid residue corresponding to position I292of SEQ ID NO:6 is GLN, or GLU; (108) the amino acid residuecorresponding to position E297 of SEQ ID NO:6 is ARG, HIS, or LEU; (109)the amino acid residue corresponding to position S299 of SEQ ID NO:6 isALA, or GLN; (110) the amino acid residue corresponding to position N300of SEQ ID NO:6 is ARG, ASP, GLN, or GLU; (111) the amino acid residuecorresponding to position K302 of SEQ ID NO:6 is ARG, GLN, GLU, ILE,LEU, MET, or PRO; (112) the amino acid residue corresponding to positionR303 of SEQ ID NO:6 is ASP, GLN, or GLU; (113) the amino acid residuecorresponding to position E307 of SEQ ID NO:6 is ARG, or GLN; (114) theamino acid residue corresponding to position D313 of SEQ ID NO:6 is PRO;(115) the amino acid residue corresponding to position Y315 of SEQ IDNO:6 is TRP; (116) the amino acid residue corresponding to position K318of SEQ ID NO:6 is ASN, or ASP; (117) the amino acid residuecorresponding to position K321 of SEQ ID NO:6 is ARG, ASP, GLU, or SER;(118) the amino acid residue corresponding to position D322 of SEQ IDNO:6 is THR; (119) the amino acid residue corresponding to position S323of SEQ ID NO:6 is ASP, or GLY; (120) the amino acid residuecorresponding to position K324 of SEQ ID NO:6 is ASP, GLU, HIS, PRO, orTRP; (121) the amino acid residue corresponding to position R325 of SEQID NO:6 is ALA, ASP, GLN, GLU, GLY, SER, or TRP; (122) the amino acidresidue corresponding to position L326 of SEQ ID NO:6 is HIS; (123) theamino acid residue corresponding to position E327 of SEQ ID NO:6 is ARG,ILE, or TYR; (124) the amino acid residue corresponding to position L328of SEQ ID NO:6 is ARG, ASP, GLU, TRP, or TYR; (125) the amino acidresidue corresponding to position W331 of SEQ ID NO:6 is GLU, LEU, orPHE; (126) the amino acid residue corresponding to position E343 of SEQID NO:6 is ASN, ASP, GLN, LEU, MET, or TYR; (127) the amino acid residuecorresponding to position E345 of SEQ ID NO:6 is ASP, or PRO; (128) theamino acid residue corresponding to position K348 of SEQ ID NO:6 is ALA,ARG, GLN, GLU, LEU, TRP, or TYR; (129) the amino acid residuecorresponding to position M349 of SEQ ID NO:6 is ARG, ASN, GLN, GLU,ILE, THR, TRP, or TYR; (130) the amino acid residue corresponding toposition N352 of SEQ ID NO:6 is ARG, GLN, GLU, LEU, or MET; (131) theamino acid residue corresponding to position K353 of SEQ ID NO:6 is ALA,ARG, GLN, GLU, HIS, or LEU; (132) the amino acid residue correspondingto position E356 of SEQ ID NO:6 is ARG, or TRP; (133) the amino acidresidue corresponding to position N357 of SEQ ID NO:6 is HIS; (134) theamino acid residue corresponding to position G361 of SEQ ID NO:6 is ASP,or PRO; (135) the amino acid residue corresponding to position D363 ofSEQ ID NO:6 is HIS, or PRO; (136) the amino acid residue correspondingto position I364 of SEQ ID NO:6 is GLU, LEU, MET, or PRO; (137) theamino acid residue corresponding to position K365 of SEQ ID NO:6 is ARG,ASN, ASP, GLU, or SER; (138) the amino acid residue corresponding toposition F366 of SEQ ID NO:6 is ARG, HIS, LEU, or TYR; (139) the aminoacid residue corresponding to position Y368 of SEQ ID NO:6 is ARG, orLEU; (140) the amino acid residue corresponding to position Q369 of SEQID NO:6 is ARG, ASP, GLU, or LEU; (141) the amino acid residuecorresponding to position Y370 of SEQ ID NO:6 is HIS; (142) the aminoacid residue corresponding to position D373 of SEQ ID NO:6 is ARG, ASN,GLU, or LEU; (143) the amino acid residue corresponding to position Y375of SEQ ID NO:6 is ALA, or TRP; (144) the amino acid residuecorresponding to position F376 of SEQ ID NO:6 is ARG, GLN, or GLU; (145)the amino acid residue corresponding to position D377 of SEQ ID NO:6 isALA, ARG, ASN, HIS, or SER; (146) the amino acid residue correspondingto position E380 of SEQ ID NO:6 is ARG, ASN, ASP, or LEU; (147) theamino acid residue corresponding to position K382 of SEQ ID NO:6 is ARG,ASN, ASP, GLN, GLU, or LEU; (148) the amino acid residue correspondingto position K384 of SEQ ID NO:6 is ARG, ASP, GLN, GLU, LEU, SER, or THR;(149) the amino acid residue corresponding to position N385 of SEQ IDNO:6 is PRO, or THR; (150) the amino acid residue corresponding toposition D386 of SEQ ID NO:6 is HIS; (151) the amino acid residuecorresponding to position R388 of SEQ ID NO:6 is GLU; (152) the aminoacid residue corresponding to position E389 of SEQ ID NO:6 is ARG, orASP; (153) the amino acid residue corresponding to position K392 of SEQID NO:6 is ARG, GLU, HIS, LEU, PHE, or TYR; (154) the amino acid residuecorresponding to position K393 of SEQ ID NO:6 is ALA, ARG, GLN, GLU,HIS, THR, TRP, or TYR; (155) the amino acid residue corresponding toposition K396 of SEQ ID NO:6 is ALA, ARG, GLN, GLU, ILE, or MET; (156)the amino acid residue corresponding to position R397 of SEQ ID NO:6 isGLN, GLU, HIS, ILE, LEU, or SER; (157) the amino acid residuecorresponding to position E400 of SEQ ID NO:6 is ARG, GLN, LEU, or TYR;(158) the amino acid residue corresponding to position S403 of SEQ IDNO:6 is ALA, ARG, GLU, LEU, PHE, THR, TRP, or TYR; (159) the amino acidresidue corresponding to position N407 of SEQ ID NO:6 is ARG, ASP, GLN,GLU, HIS, ILE, LEU, PHE, TRP, or TYR; or (160) the amino acid residuecorresponding to position L408 of SEQ ID NO:6 is ARG, ASN, ASP, GLY,THR, or TRP; or combinations thereof.

In some embodiments, the polypeptide comprises one or more ofmodifications: L3E, L3T, S4L, S4D, S4R, S4N, S4P, S4E, S4Q, S4M, K5L,K5V, K5A, K5H, K5R, K5F, K5Q, D6L, D6A, D6H, D6R, D6N, D6E, D6Q, Y7W,Y7N, Y7H, L8N, L8G, R9L, R9I, R9H, R9E, R9Q, R9T, K10D, K10A, K10R,K10N, K10P, K10E, K11I, K11V, K11H, K11Y, K11R, K11F, K11W, K11E, K11Q,S21R, S21E, S21Q, S21P, Y22W, E25R, E25I, E29L, E29R, E29Q, E29I, F30A,F30H, F30T, K32L, K32I, K32A, K32R, K32Q, K32M, E33D, E33H, E33R, E33N,E33Q, E33T, K34L, K34D, K34I, K34S, K34V, K34H, K34Y, K34R, K34F, K34Q,K34T, K34M, G35D, G35N, G35H, G35S, D36L, D36S, D36H, D36Y, D36R, D36E,D36N, D36F, D36W, D36Q, D36T, Y37E, Y37R, Y37D, Y37P, Q49W, Q49I, Q49H,Q49F, F50R, F50Q, F50H, F50S, M56W, E59R, E59S, E59T, K62R, K62A, N63R,N63E, N63D, N63Q, F64L, F64E, M66E, M66F, M66Y, K67D, K67S, K67R, K67E,K67Q, K67T, K70L, K70R, K70D, K70Q, E71R, E71Q, E71S, K72L, K72I, K72H,K72E, K72Q, K72T, E75R, E75D, E75S, E76L, E76R, E76P, D77R, D77N, D77S,K78L, K78R, K78D, K78Q, K78M, I80L, I80R, I80Q, E94L, E94R, E94Q, E94Y,S96R, S96E, S96A, T98I, T98V, T98R, T98W, T98E, T98Q, N101D, N101S,N101A, N101H, N101R, N101E, N101Q, N101T, K102Y, K102R, K102W, K102E,K102Q, K104R, K104E, K104Q, D105E, D105Q, D105I, R108I, K116L, K116I,K116H, K116Y, K116R, K116E, K116W, K116D, K116Q, K116T, P125A, D129L,D129R, D129W, D129E, D129Q, P130S, K131D, K131E, K131S, K131Y, V132S,V132Y, V132R, V132E, V132T, P134A, P134D, P134H, Y135L, Y135D, Y135R,Y135W, Y135Q, E136S, E136R, E136P, E136W, E136D, E136Q, E136T, K137L,K137I, K137V, K137R, K137W, K137E, K137Q, E140L, E140R, E140D, E140I,R141L, R141D, R141H, R141Y, R141W, R141Q, E144R, E144Q, E144D, E144I,E147R, E147Q, E147A, I148D, I148E, I148V, E150R, E150A, E151R, T152V,T152A, T152H, T152Y, T152R, T152Q, A153R, A153Q, A153G, A153S, R154S,R154P, R154E, R154Q, R154T, K155G, K155E, K155P, K155W, K155D, K155Q,K155T, Y156V, Y156R, Y156E, Y156F, Y156D, Y156Q, Y156T, Y156M, N157E,N157D, N157R, N157P, F158L, F158V, F158R, F158N, F158E, F158T, Q159L,Q159G, Q159R, Q159W, Q159E, Q159T, P160L, P160I, P160S, P160A, P160R,P160F, P160E, P160T, P160M, V161W, V161E, V161I, V161Y, V179R, V179I,S187R, S187Q, S187A, S190D, S190E, S190Y, S191L, S191A, S191H, S191Y,S191R, S191W, S191E, S191Q, S191T, K193L, K193I, K193Y, K193R, K193F,K193E, K193Q, K194L, K194D, K194A, K194R, K194E, K194Q, Y195A, Y195Q,Y195H, N197L, N197D, N197S, N197R, N197P, N197E, N197Q, P200L, P200D,P200R, P200E, P200M, N201R, N201E, W203L, W203A, W203H, W203Y, W203R,W203F, D204R, D204N, D204S, R205L, R205Q, G216R, G216N, G216T, S218T,Y219R, Y219G, D220E, D220R, D220Q, D220T, K221I, K221V, K221R, K221E,K221Q, K221T, V222I, E224Q, E224Y, E224M, D228E, D228S, K229A, K229R,K229N, K229E, K229Q, R231L, R231A, R231E, R231F, G232R, G232E, G232P,E235R, E235W, K238S, K238R, K238W, K238E, K238Q, R239S, E240V, E240H,E240Y, E240N, E240D, E240T, E240M, D241R, D241N, D241Q, D241P, R258L,R258E, E262L, E262R, E262Q, N287L, N287H, N287R, N287F, N287E, N287Q,D290R, D290E, D290Q, D290F, E291R, I292E, I292Q, E297L, E297R, E297H,S299A, S299Q, N300E, N300D, N300R, N300Q, K302L, K302I, K302R, K302P,K302E, K302Q, K302M, R303D, R303E, R303Q, E307R, E307Q, D313P, Y315W,K318D, K318N, K321E, K321D, K321R, K321S, D322T, S323D, S323G, K324D,K324H, K324P, K324W, K324E, R325D, R325S, R325A, R325G, R325W, R325E,R325Q, L326H, E327R, E327I, E327Y, L328D, L328Y, L328R, L328W, L328E,W331L, W331E, W331F, E343L, E343Y, E343N, E343D, E343Q, E343M, E345D,E345P, K348L, K348A, K348Y, K348R, K348W, K348E, K348Q, M349I, M349Y,M349R, M349N, M349W, M349E, M349Q, M349T, N352L, N352R, N352E, N352Q,N352M, K353L, K353A, K353H, K353R, K353E, K353Q, E356R, E356W, N357H,G361D, G361P, D363H, D363P, I364L, I364E, I364P, I364M, K365D, K365S,K365R, K365N, K365E, F366L, F366R, F366H, F366Y, Y368L, Y368R, Q369L,Q369R, Q369E, Q369D, Y370H, D373L, D373R, D373E, D373N, Y375W, Y375A,F376R, F376E, F376Q, D377S, D377A, D377H, D377R, D377N, E380L, E380R,E380D, E380N, K382L, K382D, K382R, K382N, K382E, K382Q, K384L, K384S,K384R, K384E, K384D, K384Q, K384T, N385P, N385T, D386H, R388E, E389R,E389D, K392L, K392H, K392Y, K392R, K392F, K392E, K393A, K393H, K393Y,K393R, K393W, K393E, K393Q, K393T, K396I, K396A, K396R, K396E, K396Q,K396M, R397L, R397I, R397S, R397H, R397E, R397Q, E400L, E400R, E400Q,E400Y, S403L, S403A, S403Y, S403R, S403F, S403W, S403E, S403T, N407L,N407D, N407I, N407H, N407Y, N407R, N407F, N407W, N407E, N407Q, L408G,L408R, L408N, L408W, L408D, or L408T; or combinations thereof.

The amino acid modifications identified herein can be made in any one ofthe SEQ ID NOs: 1-444, or active variant or fragment thereof. Thelocation of the amino acid modifications can be identified byreferencing to SEQ ID NO: 6. Amino acid sequence alignment can be madeusing known methods. FIG. 17 depicts amino acid sequence alignments ofthe native thermophilic FC4Es (SEQ ID NOs: 1-23 and SEQ ID NOs:321-373). FIG. 20 depicts the sequence alignment of all computationallydesigned stability/expression mutants from Example 13 (SEQ ID NOs46-136). FIG. 22 depicts the sequence alignment of all lysine/cysteineonly mutants from Example 14 (SEQ ID NOs 137-230). FIG. 23 depicts thesequence alignment of all lysine/cysteine mutants from Example 14 thatallowed surrounding residues to mutate (SEQ ID Nos: 232-320). In someembodiments, the polypeptides carrying one or more of the amino acidmodifications identified herein are capable of converting fructose totagatose through epimerization at the carbon-4 position of fructose.

In some embodiments, the amino acid modifications are made relative tothe native/wild-type sequences identified herein. In some embodiments,the polypeptide comprises an amino acid sequence having at least 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ IDNO:1, and the one or more modifications are relative to SEQ ID NO:1. Insome embodiments, the polypeptide has an improved D-fructoseC4-epimerase activity and/or improved stability compared to thepolypeptide of SEQ ID NO:1. In some embodiments, the polypeptide isderived from Caldithrix abyssi. In some embodiments, the polypeptidecomprises an amino acid sequence having at least 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:2, and the oneor more modifications are relative to SEQ ID NO:2. In some embodiments,the polypeptide has an improved D-fructose C4-epimerase activity and/orimproved stability compared to the polypeptide of SEQ ID NO:2. In someembodiments, the polypeptide is derived from Anaerolinea thermophila. Insome embodiments, the polypeptide comprises an amino acid sequencehaving at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequenceidentity to SEQ ID NO:3, and the one or more modifications are relativeto SEQ ID NO:3. In some embodiments, the polypeptide has an improvedD-fructose C4-epimerase activity and/or improved stability compared tothe polypeptide of SEQ ID NO:3. In some embodiments, the polypeptide isderived from Thermoanaerobacterium thermosaccharolyticum. In someembodiments, the polypeptide comprises an amino acid sequence having atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identityto SEQ ID NO:4, and the one or more modifications are relative to SEQ IDNO:4. In some embodiments, the polypeptide has an improved D-fructoseC4-epimerase activity and/or improved stability compared to thepolypeptide of SEQ ID NO:4. In some embodiments, the polypeptide isderived from Thermoanaerobacter thermohydrosulfuricus. In someembodiments, the polypeptide comprises an amino acid sequence having atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identityto SEQ ID NO:5, and the one or more modifications are relative to SEQ IDNO:5. In some embodiments, the polypeptide has an improved D-fructoseC4-epimerase activity and/or improved stability compared to thepolypeptide of SEQ ID NO:5. In some embodiments, the polypeptide isderived from Caldicellulosiruptor kronotskyensis. In some embodiments,the polypeptide comprises an amino acid sequence having at least 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ IDNO:6, and the one or more modifications are relative to SEQ ID NO:6. Insome embodiments, the polypeptide has an improved D-fructoseC4-epimerase activity and/or improved stability compared to thepolypeptide of SEQ ID NO:6. In some embodiments, the polypeptide isderived from Dictyoglomus turgidum. In some embodiments, the polypeptidecomprises an amino acid sequence having at least 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:7, and the oneor more modifications are relative to SEQ ID NO:7. In some embodiments,the polypeptide has an improved D-fructose C4-epimerase activity and/orimproved stability compared to the polypeptide of SEQ ID NO:7. In someembodiments, the polypeptide is derived from Caldilinea aerophila. Insome embodiments, the polypeptide comprises an amino acid sequencehaving at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequenceidentity to SEQ ID NO:8, and the one or more modifications are relativeto SEQ ID NO:8. In some embodiments, the polypeptide has an improvedD-fructose C4-epimerase activity and/or improved stability compared tothe polypeptide of SEQ ID NO:8. In some embodiments, the polypeptide isderived from Rhodothermus marinus. In some embodiments, the polypeptidecomprises an amino acid sequence having at least 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:9, and the oneor more modifications are relative to SEQ ID NO:9. In some embodiments,the polypeptide has an improved D-fructose C4-epimerase activity and/orimproved stability compared to the polypeptide of SEQ ID NO:9. In someembodiments, the polypeptide is derived from Methanohalobiumevestigatum. In some embodiments, the polypeptide comprises an aminoacid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or98% sequence identity to SEQ ID NO:10, and the one or more modificationsare relative to SEQ ID NO:10. In some embodiments, the polypeptide hasan improved D-fructose C4-epimerase activity and/or improved stabilitycompared to the polypeptide of SEQ ID NO:10. In some embodiments, thepolypeptide is derived from Thermoanaerobacter thermohydrosulfuricus.

In some embodiments, the polypeptide comprises an amino acid sequencehaving at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequenceidentity to SEQ ID NO:11, and the one or more modifications are relativeto SEQ ID NO:11. In some embodiments, the polypeptide has an improvedD-fructose C4-epimerase activity and/or improved stability compared tothe polypeptide of SEQ ID NO:11. In some embodiments, the polypeptide isderived from Clostridium cavendishii. In some embodiments, thepolypeptide comprises an amino acid sequence having at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:12,and the one or more modifications are relative to SEQ ID NO:12. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:12. In some embodiments, the polypeptide is derived from Kosmotogaolearia. In some embodiments, the polypeptide comprises an amino acidsequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%sequence identity to SEQ ID NO:13, and the one or more modifications arerelative to SEQ ID NO:13. In some embodiments, the polypeptide has animproved D-fructose C4-epimerase activity and/or improved stabilitycompared to the polypeptide of SEQ ID NO:13. In some embodiments, thepolypeptide is derived from Butyricicoccus pullicaecorum. In someembodiments, the polypeptide comprises an amino acid sequence having atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identityto SEQ ID NO:14, and the one or more modifications are relative to SEQID NO:14. In some embodiments, the polypeptide has an improvedD-fructose C4-epimerase activity and/or improved stability compared tothe polypeptide of SEQ ID NO:14. In some embodiments, the polypeptide isderived from Clostridium thermobutyricum. In some embodiments, thepolypeptide comprises an amino acid sequence having at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:15,and the one or more modifications are relative to SEQ ID NO:15. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:15. In some embodiments, the polypeptide is derived from Litorilineaaerophila. In some embodiments, the polypeptide comprises an amino acidsequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%sequence identity to SEQ ID NO:16, and the one or more modifications arerelative to SEQ ID NO:16. In some embodiments, the polypeptide has animproved D-fructose C4-epimerase activity and/or improved stabilitycompared to the polypeptide of SEQ ID NO:16. In some embodiments, thepolypeptide is derived from Enterobacter mori. In some embodiments, thepolypeptide comprises an amino acid sequence having at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:17,and the one or more modifications are relative to SEQ ID NO:17. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:17. In some embodiments, the polypeptide is derived from Caldisericumexile. In some embodiments, the polypeptide comprises an amino acidsequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%sequence identity to SEQ ID NO:18, and the one or more modifications arerelative to SEQ ID NO:18. In some embodiments, the polypeptide has animproved D-fructose C4-epimerase activity and/or improved stabilitycompared to the polypeptide of SEQ ID NO:18. In some embodiments, thepolypeptide is derived from Dictyoglomus thermophilum. In someembodiments, the polypeptide comprises an amino acid sequence having atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identityto SEQ ID NO:19, and the one or more modifications are relative to SEQID NO:19. In some embodiments, the polypeptide has an improvedD-fructose C4-epimerase activity and/or improved stability compared tothe polypeptide of SEQ ID NO:19. In some embodiments, the polypeptide isderived from Rhodothermus marinus. In some embodiments, the polypeptidecomprises an amino acid sequence having at least 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:20, and theone or more modifications are relative to SEQ ID NO:20. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:20.

In some embodiments, the polypeptide is derived from Rhodothermusprofundi. In some embodiments, the polypeptide comprises an amino acidsequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%sequence identity to SEQ ID NO:21, and the one or more modifications arerelative to SEQ ID NO:21. In some embodiments, the polypeptide has animproved D-fructose C4-epimerase activity and/or improved stabilitycompared to the polypeptide of SEQ ID NO:21. In some embodiments, thepolypeptide is derived from Caldibacillus debilis. In some embodiments,the polypeptide comprises an amino acid sequence having at least 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ IDNO:22, and the one or more modifications are relative to SEQ ID NO:22.In some embodiments, the polypeptide has an improved D-fructoseC4-epimerase activity and/or improved stability compared to thepolypeptide of SEQ ID NO:22. In some embodiments, the polypeptide isderived from Caloramator quimbayensis. In some embodiments, thepolypeptide comprises an amino acid sequence having at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:23,and the one or more modifications are relative to SEQ ID NO:23. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:23.

In some embodiments, the polypeptide is derived from Methanosalsumzhilinae. In some embodiments, the polypeptide comprises an amino acidsequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%sequence identity to SEQ ID NO:321, and the one or more modificationsare relative to SEQ ID NO:321. In some embodiments, the polypeptide hasan improved D-fructose C4-epimerase activity and/or improved stabilitycompared to the polypeptide of SEQ ID NO:321. In some embodiments, thepolypeptide is derived from Pseudothermotoga thermarum. In someembodiments, the polypeptide comprises an amino acid sequence having atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identityto SEQ ID NO:322, and the one or more modifications are relative to SEQID NO:322. In some embodiments, the polypeptide has an improvedD-fructose C4-epimerase activity and/or improved stability compared tothe polypeptide of SEQ ID NO:322. In some embodiments, the polypeptideis derived from Pseudothermotoga hypogea. In some embodiments, thepolypeptide comprises an amino acid sequence having at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:323,and the one or more modifications are relative to SEQ ID NO:323. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:323. In some embodiments, the polypeptide is derived fromPseudothermotoga lettingae. In some embodiments, the polypeptidecomprises an amino acid sequence having at least 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:324, and theone or more modifications are relative to SEQ ID NO:324. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:324. In some embodiments, the polypeptide is derived fromRhodothermus marinus. In some embodiments, the polypeptide comprises anamino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 98% sequence identity to SEQ ID NO:325, and the one or moremodifications are relative to SEQ ID NO:325. In some embodiments, thepolypeptide has an improved D-fructose C4-epimerase activity and/orimproved stability compared to the polypeptide of SEQ ID NO:325. In someembodiments, the polypeptide is derived from Geosporobactersubterraneus. In some embodiments, the polypeptide comprises an aminoacid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or98% sequence identity to SEQ ID NO:326, and the one or moremodifications are relative to SEQ ID NO:326. In some embodiments, thepolypeptide has an improved D-fructose C4-epimerase activity and/orimproved stability compared to the polypeptide of SEQ ID NO:326. In someembodiments, the polypeptide is derived from Melioribacter roseus. Insome embodiments, the polypeptide comprises an amino acid sequencehaving at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequenceidentity to SEQ ID NO:327, and the one or more modifications arerelative to SEQ ID NO:327. In some embodiments, the polypeptide has animproved D-fructose C4-epimerase activity and/or improved stabilitycompared to the polypeptide of SEQ ID NO:327. In some embodiments, thepolypeptide is derived from Lysinibacillus sphaericus. In someembodiments, the polypeptide comprises an amino acid sequence having atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identityto SEQ ID NO:328, and the one or more modifications are relative to SEQID NO:328. In some embodiments, the polypeptide has an improvedD-fructose C4-epimerase activity and/or improved stability compared tothe polypeptide of SEQ ID NO:328. In some embodiments, the polypeptideis derived from Clostridium stercorarium. In some embodiments, thepolypeptide comprises an amino acid sequence having at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:329,and the one or more modifications are relative to SEQ ID NO:329. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:329. In some embodiments, the polypeptide is derived from Trueperaradiovictrix. In some embodiments, the polypeptide comprises an aminoacid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or98% sequence identity to SEQ ID NO:330, and the one or moremodifications are relative to SEQ ID NO:330. In some embodiments, thepolypeptide has an improved D-fructose C4-epimerase activity and/orimproved stability compared to the polypeptide of SEQ ID NO:330. In someembodiments, the polypeptide is derived from Dictyoglomus turgidum. Insome embodiments, the polypeptide comprises an amino acid sequencehaving at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequenceidentity to SEQ ID NO:331, and the one or more modifications arerelative to SEQ ID NO:331. In some embodiments, the polypeptide has animproved D-fructose C4-epimerase activity and/or improved stabilitycompared to the polypeptide of SEQ ID NO:331. In some embodiments, thepolypeptide is derived from Caldilinea aerophila. In some embodiments,the polypeptide comprises an amino acid sequence having at least 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ IDNO:332, and the one or more modifications are relative to SEQ ID NO:332.In some embodiments, the polypeptide has an improved D-fructoseC4-epimerase activity and/or improved stability compared to thepolypeptide of SEQ ID NO:332. In some embodiments, the polypeptide isderived from Thermoflexus hugenholtzii. In some embodiments, thepolypeptide comprises an amino acid sequence having at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:333,and the one or more modifications are relative to SEQ ID NO:333. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:333. In some embodiments, the polypeptide is derived fromThermoanaerobacterium thermosaccharolyticum. In some embodiments, thepolypeptide comprises an amino acid sequence having at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:334,and the one or more modifications are relative to SEQ ID NO:334. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:334. In some embodiments, the polypeptide is derived from Petrotogamobilis. In some embodiments, the polypeptide comprises an amino acidsequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%sequence identity to SEQ ID NO:335, and the one or more modificationsare relative to SEQ ID NO:335. In some embodiments, the polypeptide hasan improved D-fructose C4-epimerase activity and/or improved stabilitycompared to the polypeptide of SEQ ID NO:335. In some embodiments, thepolypeptide is derived from Spirochaeta thermophila. In someembodiments, the polypeptide comprises an amino acid sequence having atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identityto SEQ ID NO:336, and the one or more modifications are relative to SEQID NO:336. In some embodiments, the polypeptide has an improvedD-fructose C4-epimerase activity and/or improved stability compared tothe polypeptide of SEQ ID NO:336. In some embodiments, the polypeptideis derived from Thermofilum pendens.

In some embodiments, the polypeptide comprises an amino acid sequencehaving at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequenceidentity to SEQ ID NO:337, and the one or more modifications arerelative to SEQ ID NO:337. In some embodiments, the polypeptide has animproved D-fructose C4-epimerase activity and/or improved stabilitycompared to the polypeptide of SEQ ID NO:337. In some embodiments, thepolypeptide is derived from Rhodothermus marinus. In some embodiments,the polypeptide comprises an amino acid sequence having at least 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ IDNO:338, and the one or more modifications are relative to SEQ ID NO:338.In some embodiments, the polypeptide has an improved D-fructoseC4-epimerase activity and/or improved stability compared to thepolypeptide of SEQ ID NO:338. In some embodiments, the polypeptide isderived from Dictyoglomus thermophilum. In some embodiments, thepolypeptide comprises an amino acid sequence having at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:339,and the one or more modifications are relative to SEQ ID NO:339. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:339. In some embodiments, the polypeptide is derived fromThermoanaerobacter siderophilus. In some embodiments, the polypeptidecomprises an amino acid sequence having at least 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:340, and theone or more modifications are relative to SEQ ID NO:340. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:340.

In some embodiments, the polypeptide is derived from Thermoanaerobactermathranii. In some embodiments, the polypeptide comprises an amino acidsequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%sequence identity to SEQ ID NO:341, and the one or more modificationsare relative to SEQ ID NO:341. In some embodiments, the polypeptide hasan improved D-fructose C4-epimerase activity and/or improved stabilitycompared to the polypeptide of SEQ ID NO:341. In some embodiments, thepolypeptide is derived from Thermoanaerobacter italicus. In someembodiments, the polypeptide comprises an amino acid sequence having atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identityto SEQ ID NO:342, and the one or more modifications are relative to SEQID NO:342. In some embodiments, the polypeptide has an improvedD-fructose C4-epimerase activity and/or improved stability compared tothe polypeptide of SEQ ID NO:342. In some embodiments, the polypeptideis derived from Thermoanaerobacterium thermosaccharolyticum. In someembodiments, the polypeptide comprises an amino acid sequence having atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identityto SEQ ID NO:343, and the one or more modifications are relative to SEQID NO:343. In some embodiments, the polypeptide has an improvedD-fructose C4-epimerase activity and/or improved stability compared tothe polypeptide of SEQ ID NO:343. In some embodiments, the polypeptideis derived from Thermoanaerobacterium thermosaccharolyticum. In someembodiments, the polypeptide comprises an amino acid sequence having atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identityto SEQ ID NO:344, and the one or more modifications are relative to SEQID NO:344. In some embodiments, the polypeptide has an improvedD-fructose C4-epimerase activity and/or improved stability compared tothe polypeptide of SEQ ID NO:344. In some embodiments, the polypeptideis derived from Thermoanaerobacterium thermosaccharolyticum. In someembodiments, the polypeptide comprises an amino acid sequence having atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identityto SEQ ID NO:345, and the one or more modifications are relative to SEQID NO:345. In some embodiments, the polypeptide has an improvedD-fructose C4-epimerase activity and/or improved stability compared tothe polypeptide of SEQ ID NO:345. In some embodiments, the polypeptideis derived from Thermoanaerobacterium thermosaccharolyticum. In someembodiments, the polypeptide comprises an amino acid sequence having atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identityto SEQ ID NO:346, and the one or more modifications are relative to SEQID NO:346. In some embodiments, the polypeptide has an improvedD-fructose C4-epimerase activity and/or improved stability compared tothe polypeptide of SEQ ID NO:346. In some embodiments, the polypeptideis derived from Thermoanaerobacterium xylanolyticum. In someembodiments, the polypeptide comprises an amino acid sequence having atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identityto SEQ ID NO:347, and the one or more modifications are relative to SEQID NO:347. In some embodiments, the polypeptide has an improvedD-fructose C4-epimerase activity and/or improved stability compared tothe polypeptide of SEQ ID NO:347. In some embodiments, the polypeptideis derived from Petrotoga mobilis. In some embodiments, the polypeptidecomprises an amino acid sequence having at least 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:348, and theone or more modifications are relative to SEQ ID NO:348. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:348. In some embodiments, the polypeptide is derived fromThermoanaerobacterium saccharolyticum. In some embodiments, thepolypeptide comprises an amino acid sequence having at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:349,and the one or more modifications are relative to SEQ ID NO:349. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:349. In some embodiments, the polypeptide is derived from Petrotogamobilis. In some embodiments, the polypeptide comprises an amino acidsequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%sequence identity to SEQ ID NO:350, and the one or more modificationsare relative to SEQ ID NO:350. In some embodiments, the polypeptide hasan improved D-fructose C4-epimerase activity and/or improved stabilitycompared to the polypeptide of SEQ ID NO:350. In some embodiments, thepolypeptide is derived from Spirochaeta thermophila.

In some embodiments, the polypeptide comprises an amino acid sequencehaving at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequenceidentity to SEQ ID NO:351, and the one or more modifications arerelative to SEQ ID NO:351. In some embodiments, the polypeptide has animproved D-fructose C4-epimerase activity and/or improved stabilitycompared to the polypeptide of SEQ ID NO:351. In some embodiments, thepolypeptide is derived from Ignisphaera aggregans. In some embodiments,the polypeptide comprises an amino acid sequence having at least 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ IDNO:352, and the one or more modifications are relative to SEQ ID NO:352.In some embodiments, the polypeptide has an improved D-fructoseC4-epimerase activity and/or improved stability compared to thepolypeptide of SEQ ID NO:352. In some embodiments, the polypeptide isderived from Thermotoga maritima. In some embodiments, the polypeptidecomprises an amino acid sequence having at least 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:353, and theone or more modifications are relative to SEQ ID NO:353. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:353. In some embodiments, the polypeptide is derived fromCaldanaerobacter subterraneus. In some embodiments, the polypeptidecomprises an amino acid sequence having at least 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:354, and theone or more modifications are relative to SEQ ID NO:354. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:354. In some embodiments, the polypeptide is derived from Mesotogainfera. In some embodiments, the polypeptide comprises an amino acidsequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%sequence identity to SEQ ID NO:355, and the one or more modificationsare relative to SEQ ID NO:355. In some embodiments, the polypeptide hasan improved D-fructose C4-epimerase activity and/or improved stabilitycompared to the polypeptide of SEQ ID NO:355. In some embodiments, thepolypeptide is derived from Thermosinus carboxydivorans. In someembodiments, the polypeptide comprises an amino acid sequence having atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identityto SEQ ID NO:356, and the one or more modifications are relative to SEQID NO:356. In some embodiments, the polypeptide has an improvedD-fructose C4-epimerase activity and/or improved stability compared tothe polypeptide of SEQ ID NO:356. In some embodiments, the polypeptideis derived from Halanaerobium congolense. In some embodiments, thepolypeptide comprises an amino acid sequence having at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:357,and the one or more modifications are relative to SEQ ID NO:357. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:357. In some embodiments, the polypeptide is derived fromHalanaerobium congolense. In some embodiments, the polypeptide comprisesan amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 98% sequence identity to SEQ ID NO:358, and the one or moremodifications are relative to SEQ ID NO:358. In some embodiments, thepolypeptide has an improved D-fructose C4-epimerase activity and/orimproved stability compared to the polypeptide of SEQ ID NO:358. In someembodiments, the polypeptide is derived from Halanaerobiumsaccharolyticum. In some embodiments, the polypeptide comprises an aminoacid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or98% sequence identity to SEQ ID NO:359, and the one or moremodifications are relative to SEQ ID NO:359. In some embodiments, thepolypeptide has an improved D-fructose C4-epimerase activity and/orimproved stability compared to the polypeptide of SEQ ID NO:359. In someembodiments, the polypeptide is derived from Gracilibacillus halophilus.In some embodiments, the polypeptide comprises an amino acid sequencehaving at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequenceidentity to SEQ ID NO:360, and the one or more modifications arerelative to SEQ ID NO:360. In some embodiments, the polypeptide has animproved D-fructose C4-epimerase activity and/or improved stabilitycompared to the polypeptide of SEQ ID NO:360.

In some embodiments, the polypeptide is derived from Caldanaerobactersubterraneus. In some embodiments, the polypeptide comprises an aminoacid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or98% sequence identity to SEQ ID NO:361, and the one or moremodifications are relative to SEQ ID NO:361. In some embodiments, thepolypeptide has an improved D-fructose C4-epimerase activity and/orimproved stability compared to the polypeptide of SEQ ID NO:361. In someembodiments, the polypeptide is derived from Litorilinea aerophila. Insome embodiments, the polypeptide comprises an amino acid sequencehaving at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequenceidentity to SEQ ID NO:362, and the one or more modifications arerelative to SEQ ID NO:362. In some embodiments, the polypeptide has animproved D-fructose C4-epimerase activity and/or improved stabilitycompared to the polypeptide of SEQ ID NO:362. In some embodiments, thepolypeptide is derived from Caldanaerobacter subterraneus. In someembodiments, the polypeptide comprises an amino acid sequence having atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identityto SEQ ID NO:363, and the one or more modifications are relative to SEQID NO:363. In some embodiments, the polypeptide has an improvedD-fructose C4-epimerase activity and/or improved stability compared tothe polypeptide of SEQ ID NO:363. In some embodiments, the polypeptideis derived from Caldanaerobacter subterraneus. In some embodiments, thepolypeptide comprises an amino acid sequence having at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:364,and the one or more modifications are relative to SEQ ID NO:364. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:364. In some embodiments, the polypeptide is derived fromCaldicoprobacter faecalis. In some embodiments, the polypeptidecomprises an amino acid sequence having at least 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:365, and theone or more modifications are relative to SEQ ID NO:365. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:365. In some embodiments, the polypeptide is derived fromThermoanaerobacter uzonensis. In some embodiments, the polypeptidecomprises an amino acid sequence having at least 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:366, and theone or more modifications are relative to SEQ ID NO:366. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:366. In some embodiments, the polypeptide is derived fromLactobacillus ingluviei. In some embodiments, the polypeptide comprisesan amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 98% sequence identity to SEQ ID NO:367, and the one or moremodifications are relative to SEQ ID NO:367. In some embodiments, thepolypeptide has an improved D-fructose C4-epimerase activity and/orimproved stability compared to the polypeptide of SEQ ID NO:367. In someembodiments, the polypeptide is derived from Petrotoga mexicana. In someembodiments, the polypeptide comprises an amino acid sequence having atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identityto SEQ ID NO:368, and the one or more modifications are relative to SEQID NO:368. In some embodiments, the polypeptide has an improvedD-fructose C4-epimerase activity and/or improved stability compared tothe polypeptide of SEQ ID NO:368. In some embodiments, the polypeptideis derived from Defluviitoga tunisiensis. In some embodiments, thepolypeptide comprises an amino acid sequence having at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:369,and the one or more modifications are relative to SEQ ID NO:369. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:369. In some embodiments, the polypeptide is derived from Petrotogamiotherma. In some embodiments, the polypeptide comprises an amino acidsequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%sequence identity to SEQ ID NO:370, and the one or more modificationsare relative to SEQ ID NO:370. In some embodiments, the polypeptide hasan improved D-fructose C4-epimerase activity and/or improved stabilitycompared to the polypeptide of SEQ ID NO:370. In some embodiments, thepolypeptide is derived from Petrotoga olearia. In some embodiments, thepolypeptide comprises an amino acid sequence having at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:371,and the one or more modifications are relative to SEQ ID NO:371. In someembodiments, the polypeptide has an improved D-fructose C4-epimeraseactivity and/or improved stability compared to the polypeptide of SEQ IDNO:371. In some embodiments, the polypeptide is derived fromThermophagus xiamenensis. In some embodiments, the polypeptide comprisesan amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 98% sequence identity to SEQ ID NO:372, and the one or moremodifications are relative to SEQ ID NO:372. In some embodiments, thepolypeptide has an improved D-fructose C4-epimerase activity and/orimproved stability compared to the polypeptide of SEQ ID NO:372. In someembodiments, the polypeptide is derived from Treponema caldarium. Insome embodiments, the polypeptide comprises an amino acid sequencehaving at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequenceidentity to SEQ ID NO:373, and the one or more modifications arerelative to SEQ ID NO:373. In some embodiments, the polypeptide has animproved D-fructose C4-epimerase activity and/or improved stabilitycompared to the polypeptide of SEQ ID NO:373. In some embodiments, thepolypeptide is derived from Thermofilum uzonense.

TABLE 1 Modified Polypeptides Parent Wild Mutation Position MutationPosition SEQ Plasmid Type SEQ ID (correspond to (correspond to NO. IDNO. SEQ ID NO.: 2) SEQ ID NO.: 6) 24 pA07199 2 C28A C17A 25 pA07200 2E52A E41A 26 pA07201 2 E52Q E41Q 27 pA07202 2 Y64A Y53A 28 pA07203 2Y64F Y53F 29 pA07204 2 D95A D84A 30 pA07205 2 D95N D84N 31 pA07206 2H96A H85A 32 pA07207 2 H130A H119A 33 pA07208 2 D132A D121A 34 pA07209 2D132N D121N 35 pA07210 2 E178A D167A 36 pA07211 2 E178Q D167Q 37 pA072122 E263A E245A 38 pA07213 2 E263Q E245Q 39 pA07214 2 H265A H247A 40pA07215 2 D268A D250A 41 pA07216 2 D268N D250N 42 pA07217 2 G289A G271A43 pA07218 2 G289D G271D 44 pA07219 2 Y361A Y340A 45 pA07220 2 Y361FY340F

TABLE 2 Modified Polypeptides Parent Wild Type SEQ Plasmid SEQ NO. ID IDNO. Mutation Position (correspond to SEQ ID NO.: 2) 46 pA12584 6 E75P,S96A, P97E, S113A, P160A, A188T, I189L, S191V, Y195A, N197K, V199L,G232E, L234M, G246A, A253D, N259A, N333S, S374Q 47 pA12585 6 P97E,S113A, Y135L, R143V, E147K, V182P, S191Y, V199I, G232E, G246A, G281A,L328F, S403A 48 pA12586 6 K72L, S96A, P97E, S113A, R143A, T152A, P160A,A188M, S191T, G232K, M349K 49 pA12587 6 S96A, P97E, S113A, R143V, S191T,G232D, A253S, S374Q, M383L 50 pA12588 6 S21P, E75P, S96E, P97E, S113A,I189L, S191I, G232E, I233L, G281A, M349K 51 pA12589 6 S21P, S96A, G232E,M349K, V350A 52 pA12590 6 S96A, P97E, V182P, S191T, G232E, S286A, R325E53 pA12591 6 S96A, P97E, S113A, P160E, V182P, S191I, D198K, G246A 54pA12592 6 E75P, P97E, V182P, N197K, G246A, A253S, G281A, S286V, M349K,Y372P, S403Y 55 pA12593 6 E75P, S113A, F146C, S190D, Y344H, Y372P 56pA12594 6 E147R, V182P, S191L, G281A, S374Q 57 pA12595 6 S21P, P97E,Y135L, E345P 58 pA12596 6 I202A, Y372P 59 pA12597 6 E75P, P97E, Y135P,S191L, N197E 60 pA12598 6 S113A, N333S 61 pA12599 6 R143A, V182P, Y344H,E345P 62 pA12600 6 S54T, S96A, R143A, A188M, S191V, G208A, G232E, E240T,A253S, R266V, S286A, R325E, I330L, E345P, F376R 63 pA12601 6 S54T, S96A,P97E, N101E, S113A, R143A, G232E, G246A, G281A, E295P, R296E, F358L,E360D, M383L 64 pA12602 6 K34R, S54T, S96A, S113A, A188T, N197K, G246A,N287D, V350A 65 pA12603 6 S21P, S54T, S96A, S113A, R143A, S191I, M349R66 pA12604 6 K34R, P97E, S113A, G232E 67 pA12605 6 S54T, E75P, S96A,P97E, S113A, L126C, S127A, R143V, V182P, A188T, S191T, N197K, V199H,G208A, G232E, G246C, A253S, R266V, G281A, N287D, E345P, V350A, E360D,V362I, F376R 68 pA12606 6 S54T, E75P, S96A, S113A, E140I, E147R, A188M,S191T, N259W, E360D, S374A, N385P 69 pA12607 6 S21P, S54T, S96A, P97E,V182P, G208A, E240T, S374C 70 pA12608 6 S21P, S27A, S54T, K78R, P97D,T98E, N101E, S113A, L126C, S127A, E147R, V182P, A188T, S191T, N197E,V199H, I202M, I215V, Y219H, G232D, E240T, A273W, G281A, Y344H, E345P,V350A, D363P, S374C 71 pA12609 6 S27A, K34D, Y37P, S54T, E75P, S113A,L126C, S127A, F146C, Y219H, N259W, R266V, N333S, E345P, V350A, D363P 72pA12610 6 S21P, S113A, S127A, E151R, A188T, G232D, E240T, Y344H, V350A,F358L, F366L 73 pA12611 6 S21P, Y37P, T98E, F146C, A188T, Y219H, N259W,D363P 74 pA12612 6 Y22W, S54T, L126C, E151R, V182P, A188T, Y219H, G232E,E345P, V350A, F358L 75 pA12613 6 P97E, N101E, S113A, S127A, V199H,Y219H, E240T, N259W, Y344H 76 pA12614 6 S27A, S54T, E75P, T98E, N101E,S113A, A188T, E240T, E297G, V350A 77 pA12615 6 Y14F, P97E, F110Y, Y115F,Y135M, R143A, E147K, S191Y, G246A, G281A, L328F, M349K, S403A 78 pA126166 P97E, S113A, V182P, A188F, G232E, V237L, A253S, M349T, M383L 79pA12617 6 S96A, P97E, S113A, E147K, P160A, V182A, S191I, G232D, G246A,S286A, R325E, M349K 80 pA12618 6 S21P, E75P, R143A, I148V, S191I, V244L,E345P 81 pA12619 6 S54T, K72L, P97E, A188M, S191T, D198K, G232E, R325E,S403A 82 pA12620 6 S19N, K72V, S96A, P97E, S190D, S191T, N197K, G232E,G246A, G281A, Y372P, S403Y 83 pA12621 6 S21K, E75P, P97E, S113A, R143V,S191T, V199I, G208A, G232E, V244L, G246A, N259A, G281A, S286A, E345P,Y372P, S374Q 84 pA12622 6 S27A, E75P, Y135L, E147R, V182P, S190E, S191T,E240T, Y344H, M349K, S374Q 85 pA12623 6 S21P, S27A, P97E, S113A, Y135T,L192F, Y219D, G281A, S286A, Y344H, E345P, F376E 86 pA12624 6 S27A, E75P,S96A, G232D, S286A, E345P, Y372P 87 pA12625 6 S27A, N101E, S113A, Y135P,S187E, S190E, Y344H 88 pA12626 6 S96A, Y135L, E151R, V182P, S190D,G232D, K365R 89 pA12627 6 S27A, E75P, S187R, G281A 90 pA12628 6 S27A,S54T, S96A, F146C, V182P, Y344H, V350A, S403A 91 pA12629 6 S21P, Y22W,S54T, E75P, S113A, V182P, N197E, E240T, G246V, S286A, R325E, I330L,Y344H, E345P, F376R 92 pA12630 6 S21P, E75P, S96A, N101E, S127A, A188T,S191T, N197E, V199H, N201R, G208A, G232E, E240T, G246C, S286A, N287D,R296E, N333A, M349R, S374C 93 pA12631 6 S27A, S113A, E140I, R143A,V182P, A188M, S191T, G232E, L242W, G246A, A253P, N259W, G281A, R325E,N333A, V350A, E360D, R379C, N385P 94 pA12632 6 E75P, S96A, P97E, S127A,R143V, V182P, S191T, V199C, V237L, Y344H, M349A, F358L 95 pA12633 6S21P, S27A, P97E, S113A, S127A, T152A, A188M, S191V, G232E, R266V,A273W, S286A, N287D, N333A, E345P, M349K, V362I 96 pA12634 6 S54T, E75P,S96A, P97E, A188T, L213M, G232E, Y344H, S374C 97 pA12635 6 S54T, P97E,S127A, R143A, E345P, M349K, V350A 98 pA12636 6 Y22W, K34D, Y37P, S54T,E75P, S113A, F146C, A188T, G232D, R266A, S286A, R296E, Y344H, E345P,K393H 99 pA12637 6 S21P, Y22W, S27A, D36T, S54T, E75P, P97E, T98E,S127A, E147R, S191L, N197E, V199H, G232E, D241P, Y255E, N287D, Y344H,V350A, K365T, S374C 100 pA12638 6 S21P, S27A, E75P, P97E, N101E, S113A,E147K, V182P, A188T, S190E, Y219H, Y255D, G281A, E343N, Y344H, V350A,E360D, K393H 101 pA12639 6 Y22W, D36S, E75P, P97D, N101E, S113A, L126C,S127A, V182P, N197E, Y219H, D241P, A253S, Y255D, S286A, R296E, E343N,E345P, K365T, F376R 102 pA12640 6 S54T, K62R, P97E, S113A, G232E, E240T,N259W, S286A, E297G, F376H 103 pA12641 6 V13I, S21P, S27A, S54T, E75P,P97E, T98E, L126C, F146C, E147R, A188T, S190E, I202A, D241P, N259W,G281A, R296E, R303E, W331H, E345P, M349A, V350A, E360D, S374A, F376H 104pA12642 6 S27A, S96A, T98E, N101E, L126C, Y219H, E240T, A253T, R296Y,Y344H, E345P, M349A, F366L 105 pA12643 6 S27A, G35D, K72L, E75P, P97E,F110Y, S113A, Y135M, E147K, S191L, N197K, G232E, G281A, L284K, I292L,L328F, M349K, Y372P, F376K 106 pA12644 6 P97E, Y135K, R143V, G232D,V237L, S286A, D290K, Y344H, M349T, M383L 107 pA12645 6 S27A, K72L, E75P,S191V, L192F, N197K, V199L, I211V, G232E, S286A, S374Q 108 pA12646 6S21P, S27A, S54T, K72V, K78R, P97E, S190D, S191I, D198K, S286A, R325E,M349K 109 pA12647 6 S27A, S113A, Y135L, A188M, S190D, S191T, N197E,R325E, F376Q 110 pA12648 6 S27A, N101E, S113A, Y135L, E147K, I148V,L192F, N197K, E240T, A253D, Y255K, N259D, G281A, S286A, D290K, E345P,M349C, K365R, S374Q 111 pA12649 6 S21P, S27A, S54T, E75P, P97E, N101E,Y135L, S187E, S190E, S191Y, N197E, V199L, G232D, A253D, R296D, Y344H,E345P, K365R 112 pA12650 6 S21E, S54T, S96A, D105E, S113A, Y135L, V182P,S190E, S191A, E240T, A253T, S286A, K365R 113 pA12651 6 S21E, S54T, E75P,N101E, S113A, Y135M, E147K, S190E, G232P, Y344H, M349K, K365R, S403A 114pA12652 6 S54T, E75P, P97E, N101E, S113A, Y135T, I148V, S187E, S190E,L192F, Y219D, G232D, L234A, S286A, E297K, E327R, V350A, K353R 115pA12653 6 S27A, S54T, E75P, P97E, T98E, N101A, S113A, S127A, S191V,N197K, V199L, L213M, G232E, G246A, N259W, S286A, N287D, K321H, N333S,Y344H, E345P, M349A, V350A 116 pA12654 6 S21P, S27C, S54T, E75P, T98E,S113A, S127A, V182P, A188F, S191V, G232E, A253S, Y255E, S286D, D290K,E291D, N333S, Y344H, V350A, F358L, V362I, F366L, S374T 117 pA12655 6S21P, G35D, S54T, P97E, Y135Q, R143A, A188T, N197K, G232E, G246A, G281A,N287D, E295P, L328F, N333A, M349A, Y372P 118 pA12656 6 S21P, E75P, P97E,T98E, N101A, R143V, V182P, G208A, E240T, G246V, A253S, R266V, S286A,K321H, R325D, Y344H, F366L 119 pA12657 6 Y22W, S27A, S54T, E75P, S96A,P97E, N101A, S113A, S127A, R143A, G232E, E240T, V244L, S286D, M349R,V350A 120 pA12658 6 S21P, D36S, S54T, N63R, E75P, D93H, P97D, N101E,Y115F, L126C, S127A, Y135N, E147R, V182P, S187R, A188T, V199H, Y219H,G246A, Y255E, N259W, R266A, G281A, N287D, K324D, W331H, N333S, Y344H,M349A, E360D, M383L, K393H, S403A 121 pA12659 6 Y22W, S27A, K62R, N63R,S96A, P97E, T98E, D105E, S113A, L126C, S127A, E151R, V182P, S187K,S190E, S191A, N197A, G232E, E240T, Y255D, S286A, R296Y, E297R, R303E,K324D, E343N, E345P, M349A, V350A, F358L, F366L 122 pA12660 6 V13I,S21P, K34D, Y37P, N63R, M66Y, E75P, P97D, T98E, L126C, V179I, S190E,S191L, L192H, N197K, Y219H, G232D, N259W, R266A, E291D, R303E, K324D,N333S, Y344H, V350A, D363P, M383L, K393H 123 pA12661 6 S21P, Y22W, S27A,S54T, E75P, P97E, R143A, E144R, A188T, S191A, V199H, Y219H, V230A,N259W, S286A, N287D, K324E, E327R, W331H, Y344H, E345P, M349E, K353R,E360D, I364W, F376H 124 pA12662 6 S21P, Y22W, Y37P, S54T, M66Y, D77N,P97E, S127A, A188T, S190E, S191A, Y219H, G232E, E240T, E262Q, R266A,S286A, R296Y, N333S, Y344H, E345P, M349A, V350A, D363P, K365T, S403A 125pA12663 6 S21P, S54T, S96A, P97E, N101K, S113A, Y135K, R143V, R154Q,V182K, A188F, S191T, N197D, G232E, V237L, A253D, Y255Q, N259R, V282I,S286A, D290Q, I292L, K302L, Y344H, M349K, S374Q, M383L 126 pA12664 6Y14F, S21K, S27A, K34M, G35H, K72L, E75P, S96A, F146C, P160A, S187T,Y195A, N197K, V199L, N201K, Y255K, A256L, G281A, K302L, Y344H, S374Q 127pA12665 6 S27A, G35D, S54T, E75P, S96A, Y115F, E147K, I148V, T152A,P160A, A188M, S190E, S191V, V199I, M349K 128 pA12666 6 K34M, Y37P, S54T,M66F, E75P, E76P, S96A, I148V, T152A, V182P, S190D, S191Y, L192F, N197K,I202A, D241G, A253D, N259E, S286A, R296D, E297K, K324E, E327R, N333S,Y344H, E345P, V350A, Y372P 129 pA12667 6 S27A, S54T, M66F, E75P, E76P,P97E, D105E, S113A, Y135D, T142E, E147K, V179I, S187R, A188F, S190E,G232P, E240T, A253D, Y255Q, G281A, S286A, D290N, K324E, E327R, L334Y,E345P, M349K 130 pA12668 6 M66F, S96A, T98Q, N101E, D105E, Y135P, R154Q,V182P, D184E, S187N, A188M, S190E, L192F, N197D, S218G, G232D, E240T,A253S, Y255K, D290N, E297K, E327R, E345P, M349A, K365R 131 pA12669 6S15A, S21P, G35D, S54T, E75P, S96A, T98E, N101A, S113A, K131A, V182P,S191T, G232E, E240T, G246A, N259W, R266V, G281A, N287D, D290A, K321T,K324D, M349K, E360D, V362I, S374A, F376H, R379C, N385P, S403A 132pA12670 6 S21P, K34Y, S54T, N63R, K78R, S96A, P97E, N101A, S113A, E147K,V182P, S190E, S191V, N197E, V199H, I202A, G208A, G232E, G246C, Y255E,G281A, N287D, D290K, E297H, K324D, M349A, S374C, R397K 133 pA12671 6S21P, S27A, K62R, E75P, P97E, S113A, S127A, D129E, R143A, V179I, S191T,V199H, N201R, Y219H, E240T, G246V, A253P, Y255E, R266V, N287D, R296E,M349A, N352E, Y372P, D373A, S374C 134 pA12672 6 Y22W, K62R, N63R, M66Y,K67Q, E75P, D93H, S96A, N101A, F110Y, Y115F, L126C, D128G, R143A, E144R,I148V, E151R, S187K, S190E, V199H, I202A, I215V, G232E, D241P, R266A,G281A, S286A, N287D, D290K, E297H, R303E, K321R, K324E, R325N, E327R,N333S, Y344H, E345P, S374C, F376H, D377A 135 pA12673 6 V13I, S21P, Y22W,S27A, F30H, K34D, S54T, N63R, E75P, D93H, S96A, P97E, T98E, N101E,D105E, F110Y, Y115F, K131A, E136D, R143A, F146C, E147R, I148V, S187K,L192F, N197Q, N201Q, D204N, G232D, D241P, A253N, Y255D, R280E, S286A,N287D, R296E, E297Q, R325Q, E327R, L328W, N333S, E343N, V350A, N352E,D363P, S374A, M383L 136 pA12674 6 V13I, I16V, S21P, Y22W, S27A, D36S,M66Y, D77N, D93H, T98E, N101A, L126C, S127A, D128G, K131A, Y135Q, F146C,V182P, S187K, L192F, V199H, P200G, V230A, G232E, E240T, Y255D, G281A,E297Q, R303E, R325N, W331H, N333S, E343N, Y344H, N352E, V362I, I364W,F376R, S403H 137 pA14284 6 K5A, K10N, K116R, K238S, K302Q, K318D, K321D,K324D, K348R, K353R, K365R, K382R 138 pA14285 6 K11L, K32L, K34M, K67S,K70Q, K193Q, K318D, K321D, K324E, K348L, K353L 139 pA14286 6 K10N, K34E,K70R, K131E, K137E, K155E, K321D, K324D, K348R, K382R, K384D 140 pA142876 K5F, K10M, K11L, K62V, K70R, K116I, K131E, K194A, K321D, K324D, K353L,K384S 141 pA14288 6 K11L, K70Q, K78L, K137L, K238S, K321D, K3655 142pA14289 6 K10N, K70R, K104R, K155E, K193A, K238S, K321D, K353L, K382R,K384T 143 pA14290 6 K5L, C17G, K34M, K72L, K78L, K102Y, K193Q, K321D,K324E 144 pA14291 6 K10N, C17T, K34T, K62R, K72L, K116H, C122T, K131Q,K137T, K193E, K194E, K321D, K324D, K382R 145 pA14292 6 C17T, K34Q, K62R,K70R, K72V, K131Q, K155R, K194T, K229R, K238S, K365R 146 pA14293 6 K10R,K131Q, K229T, K321D, K348R, K353R, K382R 147 pA14294 6 K67T, K78L,K102Y, K131E, K365R 148 pA14295 6 K10N, K11L, C17T, K32M, K34L, K137L,K384S 149 pA14296 6 K10N, C17T, K32M, K34L, K70R, K102L, K131Y, K137L,K193Q, K221V, K229R, K238S, K321D, K382R, K393H 150 pA14297 6 K5W, K10R,K32M, K67Q, K70R, K78M, K131Q, K193I, K348L 151 pA14298 6 K70R, K104N,K117L, K221R, K229T, K348R, K353Q, K384D 152 pA14299 6 K11Q, K34Q, K62R,K67T, K104Q, K131E, K137T, K318D, K321D, K353R, K384E 153 pA14300 6K78L, K229R, K302I, K365S 154 pA14301 6 K5M, K10L, K67T, K70R, K72L,K131Q, K137L, K221R, K318D, K353W, K365H, K384D, K392R, K396M 155pA14302 6 C17T, K67T, K70R, K102R, K104N, K155N, K318D, K321D 156pA14303 6 K104R, K131R, K193E, K238S, K348L 157 pA14304 6 K5H, K67T,K78L, K117W, C122A, K155N, K302L, K318D, K353L, K384S, K396L 158 pA143056 K62R, K67T, K104N, K131Q, K137Q, K221R, K238S, K324S, K382R 159pA14306 6 K5L, K10N, K11Q, K62R, K67T, K70R, K131Q, K137Q, K193E, K194E,K221R, K302R, K321D, K324D, K348R, K382R, K384D 160 pA14307 6 K10N,C17T, K70R, K104R, C122A, K131E, K137E, K155E, K193E, K229R, K321D,K324D, K348R, K353R 161 pA14308 6 K5H, K11V, K72L, K116T, C122T, K324E,K382L, K384E, K393Y 162 pA14309 6 K5F, K10N, K11Q, C17A, K32Q, K34Q,K67T, K72L, K137T, K229R, K238S, K318D, K321D, K324D, K348R, K353Q 163pA14310 6 K5F, K34Q, K72T, K78Q, K137T, K155N, K221T, K238S, K302R,K324D, K382R 164 pA14311 6 K62V, K70R, K78L, K104Q, K116I, K155N, K193Q,K221V, K229R, K321D, K324D, K348L, K382R, K393H 165 pA14312 6 K5W, K10R,C17T, K62R, K67T, K70R, K72L, K78R, K104R, K116R, K137E, K193R, K194T,K238S, K302Q, K321D, K348R, K353R, K365R, K384E 166 pA14313 6 K10L,K11Y, K34T, K62V, K67T, K102Y, K116Y, K131Q, K137L, K155Y, K193A, K194A,K221R, K238S, K318D, K353W, K382R, K384L 167 pA14314 6 K5A, K11Q, C17T,K32Q, K34E, K67T, K70R, K72L, K102R, K104R, K131E, K155E, K229R, K238S,K302Q, K318D, K321D, K324D, K353R, K384T 168 pA14315 6 K5A, K10N, K67T,K70R, K78Q, K102R, K131E, K137E, K155E, K193E, K194T, K229R, K321D,K348R, K353R, K365R 169 pA14316 6 K5F, K10N, K11Q, C17I, K34Q, K62R,K67T, K70R, K104Q, C122T, K131E, K221T, K238S, K269I, K318D, K321D,K324D, K348R, K353Q, K382R, K384E 170 pA14317 6 K5A, K32I, K62Y, K67T,K72L, K116L, K193A, K194T, K221I, K229R, K238S, K302I, K318D, K321D,K353L, K382R, K384T 171 pA14318 6 K70R, K78L, K102Y, K131E, K269L,K348V, K365R, K392M, K393L 172 pA14319 6 K5F, K11Q, K32Q, K62R, K78Q,K116Q, K137T, K229R, K318D, K321D, K324D, K348R, K353Q, K384D 173pA14320 6 K5F, K10M, K34L, K67T, K70R, K78L, K104R, K131E, K193Q, K194R,K221V, K238W, K324D, K353W, K365S, K384S 174 pA14321 6 K11Y, K34L, K70R,K221I, K229R, K238S, K318D, K324D, K348V, K365R, K393L 175 pA14322 6K32I, K62V, K104I, C122T, K137L, K194A, K238S, K318D, K353L, K365S,K384E, K392L 176 pA14323 6 K11V, K32L, K34Y, K62V, K67R, K70Q, K78R,K102L, K116T, K131R, K155E, K194A, K221I, K318D, K321D, K348L, K365R,K382L 177 pA14324 6 K32L, K34M, K67S, K78Q, K102L, K116T, K194S, K302I,K321D, K348L, K353L, K382L 178 pA14325 6 K10L, K11Y, C17T, K62Y, K67T,K70R, K72L, K117M, C122V, K131Q, K137L, K155Y, K193A, K221R, K229T,K269V, K318D, K321D, K324D, K353W, K365H, K382R, K392L, K396M 179pA14326 6 K10N, K11V, C17G, K32L, K67S, K78L, K193E, K302I, K318D,K353L, K365R, K384T, K392L 180 pA14327 6 K10N, K11L, K32L, K78Q, K104R,K116T, K131E, K194R, K318D, K321D, K348L, K353L, K365S, K382L, K384E 181pA14328 6 K5L, K72L, K78L, K137L, K155E, K238S, K302L, K321D, K365S,K393Y 182 pA14329 6 K5M, K10L, K11L, C17T, K32M, K34L, K70R, K72L, K78L,K102L, K116I, K155N, K194S, K221V, K238S, K318D, K321D, K324D, K348L,K382R 183 pA14330 6 K10N, K11L, K32M, K34L, K62V, K67T, K78L, K102L,K116I, K131Y, K137L, K155N, K193Q, K194R, K229R, K302L, K318D, K321D,K324D, K348L, K353W, K382R, K384S 184 pA14331 6 C17G, K34M, K70Q, K104R,K131R, K302I, K348L, K365R, K384Q, K393Y 185 pA14332 6 K5L, C17G, K34T,K67S, K72L, K78L, K269N, K321D, K382L, K393Y 186 pA14333 6 K5W, K10V,K11Y, K32M, K34T, K62V, K67T, K70R, K78L, K102Y, K104F, K116Y, K131Q,K137L, K155Y, K193A, K194A, K221R, K229S, K238W, K302L, K318D, K321D,K324D, K348L, K353W, K365H, K382R, K384D 187 pA14334 6 K5W, K11Y, K32M,K34T, K70R, K72L, K78M, K102Y, K104F, K116Y, C122V, K131Q, K193A, K194A,K238W, K269L, K302L, K318D, K321D, K324D, K348L, K382R, K384D, K392L,K393Y 188 pA14335 6 K5A, K10N, K11Q, C17L, K32Q, K34E, K67T, K70R, K72L,K78Q, K102R, K104R, K116R, K117W, C122T, K131E, K155E, K194R, K229R,K238S, K269L, K302R, K321D, K365R, K382R, K384D 189 pA14336 6 K5L, K10A,C17G, K32L, K34Y, K70Q, K72L, K116T, K131R, K155E, K194A, K221I, K238S,K302I, K318D, K321D, K324E, K348L, K353L, K384Q, K396M 190 pA14337 6K10N, K11L, C17I, K32M, K34L, K62Y, K67T, K72T, K78L, K116L, K117W,C122A, K131E, K155N, K221V, K229R, K269I, K302M, K321D, K324D, K348L,K353W, K382R, K384R, K392L, K396R 191 pA14338 6 K11Q, K34E, K62R, K72L,K104R, C122A, K137E, K193E, K221Q, K229R, K318D, K321D, K324D, K348R,K353R, K365R, K384D 192 pA14339 6 K5A, K10L, K11Q, K32I, K62V, K70R,K72L, K78L, K102Y, K104R, K116L, K131E, K137L, K155E, K229R, K318D,K321D, K348V, K353L, K382R, K384T, K392R 193 pA14340 6 K5M, K11Q, C17S,K34L, K62V, K67T, K70R, K72L, K102Y, K104Q, K131Y, K137L, K193Q, K221V,K229R, K238W, K302L, K318D, K321D, K353L, K365S, K384R, K392L, K393H,K3961 194 pA14341 6 K5H, K10N, K32L, K34T, K67S, K70Q, K78L, K102L,K104R, K116T, K131E, K137L, K194R, K229R, K238S, K318D, K321D, K365R,K382L 195 pA14342 6 K5F, K10L, K11L, K32M, K34L, K62V, K67T, K70R, K78L,K102Y, K104Q, K116I, K131E, K137L, K155N, K193Q, K194A, K221I, K229R,K238W, K302L, K318D, K321D, K324D, K348L, K353W, K365S, K382R, K384D 196pA14343 6 K5L, K11V, K32L, K34M, K62V, K67S, K70Q, K78L, K155R, K194A,K229R, K302L, K321D, K324E, K348L, K365R, K384Q, K393Y 197 pA14344 6K5W, K10N, K11Q, C17T, K34T, K62R, K67T, K72L, K102R, K131Q, K137Q,K155H, K194E, K221R, K229T, K302Q, K318D, K321D, K353R, K382R 198pA14345 6 K5L, K11L, K32I, K34T, K62V, K67S, K70Q, K72L, K116T, K131E,K137L, K155E, K193E, K229A, K324E, K348L, K353L, K382L, K384R, K392L,K393Y 199 pA14346 6 K11L, K67S, K72L, K104I, C122T, K131R, K193E, K194A,K221I, K229A, K302L, K318D, K321D, K324E, K348L, K353L, K365S, K382L,K392L, K396M 200 pA14347 6 K5H, K11V, C17G, K32I, K34T, K70Q, K72L,K78L, K104R, K117L, K131E, K155E, K194T, K221I, K229R, K269N, K321D,K348L, K353L, K365R, K382L, K393Y, K396I 201 pA14348 6 K5F, K10M, K11L,C17I, K32M, K34L, K62V, K67T, K70R, K72L, K78L, K102L, K104Q, K116I,K117V, C122T, K131E, K137E, K155N, K193Q, K194A, K221V, K229R, K238S,K269I, K302L, K318D, K321D, K324D, K348L, K353L, K365S, K382R, K384R,K392L, K393H, K396M 202 pA14349 6 K5W, K10N, K11Q, C17I, K32Q, K34T,K62R, K67T, K70R, K72L, K78R, K102R, K104R, K116H, K117V, C122V, K131Q,K137T, K155H, K193E, K194E, K221R, K229T, K238R, K269V, K302Q, K318D,K321D, K324D, K348R, K353R, K365H, K382R, K384D 203 pA14350 6 K5A, K10N,K11Q, C17T, K32Q, K34E, K62R, K67T, K70R, K72L, K78Q, K102R, K104R,K116R, K131E, K137E, K155E, K193E, K194T, K221Q, K229R, K238S, K302Q,K318D, K321D, K324D, K348R, K353R, K365R, K382R, K384D 204 pA14351 6K5L, C17G, K32I, K34M, K62V, K72L, K78L, K102L, K116T, C122T, K131E,K137L, K155E, K193Q, K194R, K229R, K269N, K302I, K318D, K321D, K324E,K348L, K353L, K365S, K384R, K392L, K393Y 205 pA14352 6 K5F, K10N, K11Q,K32Q, K34Q, K62R, K67T, K70R, K78Q, K102R, K104Q, K116Q, K131E, K137T,K155N, K193Q, K194S, K221T, K229R, K238S, K302Q, K318D, K321D, K324D,K348R, K353R, K365S, K382R, K384E 206 pA14353 6 K5L, K10N, K11E, C17V,K32Q, K34H, K62R, K67Q, K70R, K72L, K78R, K102R, K104R, K116R, K131R,K137E, K155E, K193E, K194T, K221R, K229N, K238S, K302R, K318D, K321D,K324S, K348R, K353Q, K365R, K382N, K384Q 207 pA14354 6 K5L, K10N, K11V,C17I, K32S, K34F, K62V, K67Q, K70R, K72I, K78M, K102L, K104F, K116W,K117V, C122A, K131R, K137E, K155E, K193A, K194A, K221I, K229Q, K238S,K269V, K302L, K318D, K321D, K324S, K348L, K353L, K365R, K382N, K384R,K392L, K393F, K396F 208 pA14355 6 K5L, K10N, K11L, K32L, K34M, K62V,K67S, K70Q, K78L, K102Y, K104R, K116T, K131E, K137L, K155E, K193Q,K194R, K221I, K229R, K238S, K302I, K318D, K321D, K324E, K348L, K353L,K365S, K382L, K384E 209 pA14356 6 K5L, K10A, K11L, K32I, K34T, K62V,K67S, K70Q, K78L, K102Y, K104I, K116T, K131E, K137L, K155E, K193E,K194A, K221I, K229A, K238S, K302L, K318D, K321D, K324E, K348L, K353L,K365S, K382L, K384E 210 pA14357 6 K5W, K10R, K11R, K32Q, K34Q, K62R,K67T, K70R, K78Q, K102R, K104R, K116R, K131Q, K137E, K155D, K193R,K194T, K221R, K229T, K238S, K302Q, K318E, K321D, K324D, K348R, K353R,K365R, K382R, K384E 211 pA14358 6 K5M, K10M, K11Q, C17S, K32M, K34L,K62V, K67T, K70R, K72L, K78M, K102L, K104R, K116I, K131Y, K137L, K155N,K193A, K194S, K221I, K229R, K238S, K302L, K318D, K321D, 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K396I 216 pA14363 6 K5A, K10N, K11V, C17G, K32I,K34M, K62V, K67S, K70Q, K72L, K78R, K102Y, K104I, K116S, K117L, C122T,K131E, K137L, K155E, K193E, K194A, K221I, K229A, K238S, K269N, K302L,K318D, K321D, K324E, K348R, K353L, K365S, K382L, K384R, K392L, K393Y,K3961 217 pA14364 6 K5L, K10R, K11V, C17G, K32I, K34M, K62V, K67S, K70Q,K72L, K78L, K102Y, K104R, K116T, K117L, C122T, K131R, K137L, K155R,K193I, K194A, K221I, K229R, K238S, K269N, K302L, K318D, K321D, K324E,K348L, K353L, K365R, K382L, K384R, K392L, K393Y, K3961 218 pA14365 6K5R, K10R, K11R, K32R, K34R, K62R, K67R, K70R, K78R, K102R, K104R,K116R, K131R, K137R, K155R, K193R, K194R, K221R, K229R, K238R, K302R,K318R, K321R, K324R, K348R, K353R, K365R, K382R, K384R 219 pA14366 6K5R, K10R, K11R, C17R, K32R, K34R, K62R, K67R, K70R, K72R, K78R, K102R,K104R, K116R, K131R, K137R, K155R, K193R, K194R, K221R, K229R, K238R,K302R, K318R, K321R, K324R, K348R, K353R, K365R, K382R, K384R, K392R,K393R, K396R 220 pA14367 6 K5R, K10R, K11R, C17R, K32R, K34R, K62R,K67R, K70R, K72R, K78R, K102R, K104R, K116R, K117R, C122R, K131R, K137R,K155R, K193R, K194R, K221R, K229R, K238R, K269R, K302R, K318R, K321R,K324R, K348R, K353R, K365R, K382R, K384R, K392R, K393R, K396R 221pA14368 6 K102Y, K131Q, K221R, K318D, K321D 222 pA14369 6 K102L, K131E,K221I, K318D, K321D 223 pA14370 6 K102R, K131E, K221T, K318D, K321D 224pA14371 6 C17T, K102R, K131Q, K221R, K269V, K318D, K321D 225 pA14372 6C17L, K102L, K131E, K221R, K269L, K318D, K321D 226 pA14373 6 C17G,K102Y, K131E, K221I, K318D, K321D 227 pA14374 6 C17S, K102L, K131E,K221V, K318D, K321D 228 pA14375 6 C17T, K102R, K131E, K221Q, K318D,K321D 229 pA14376 6 C17G, K102L, K131E, K221Q, K269N, K318D, K321D 230pA14377 6 C17G, K269N 231 pA14378 6 Y53F, D167A, Y340A 232 pA15875 6L3E, S4R, K5R, D6E, Y7N, L8G, R9Q, K10R, K11E, S21E, E25R, E29R, K32R,K34R, G35H, Y37R, Q49H, F50R, K62R, N63R, F64E, M66E, K67R, K70R, K72E,E75D, D77R, K78R, E94R, S96R, T98E, K102E, K104R, D105Q, K116D, D129E,K131E, P134D, E136R, 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E380D, K382R,K384R, K392R, K393E, K396R, S403E, N407D, L408G 234 pA15877 6 S4R, D6Q,Y7N, L8N, R9E, K10P, K11V, S21P, K32M, K34L, G35S, Q49F, F50Q, K62R,N63R, K67E, K70R, E71S, E75D, D77R, K78M, E94L, S96E, T98Q, N101E,K102Y, K104R, D105Q, K116Q, K131E, V132T, P134D, Y135W, E136R, K137R,E151R, A153S, R154P, K155P, Y156V, N157P, F158N, Q159T, P160I, S190E,S191T, K193E, K194R, N197E, W203R, G216N, D220R, K221T, K229Q, R231E,G232E, E235R, K238E, E240T, R258E, E262R, N287Q, D290R, E297H, N300D,K302R, D313P, K318D, K321D, K324P, R325Q, L328E, E345P, K348E, M349E,N352R, K353Q, I364E, K365R, E380N, K382N, K384D, N385T, S403A, N407Q 235pA15878 6 S4R, K5Q, D6R, Y7N, L8G, R9E, K10P, K11V, S21E, K32Q, K34S,G35N, D36T, Q49H, F50R, E59R, K62R, N63R, M66E, K67E, K70R, E71S, E75D,D77R, K78R, E94R, S96E, T98E, N101Q, K102E, K104R, D105E, K116R, D129Q,P130S, K131E, V132T, P134D, E136R, K137E, E140R, R141Q, E147R, I148E,R154T, K155T, Y156D, N157P, F158N, Q159E, P160T, V179R, S190E, S191E,K193E, K194R, N197R, P200D, W203R, G216T, Y219R, K221R, K229Q, G232E,K238R, E240T, N287R, D290R, E297H, K302E, R303E, E307R, K318N, K321E,K324D, R325Q, E327R, L328E, E343D, E345P, K348R, M349E, N352R, K353Q,I364E, K365R, Q369R, E380D, K382R, K384Q, S403R, N407E 236 pA15879 6S4R, K5H, D6Q, Y7N, L8N, R9E, K10P, K11V, S21P, K32Q, K34S, G35N, D36S,F50H, N63E, K67R, K70R, E71S, E75D, D77R, K78R, E94Q, S96E, T98E, N101Q,K102E, K104R, D105E, K116L, D129Q, K131E, V132T, P134D, E136R, K137E,E140R, R141H, E147Q, A153S, R154P, K155P, Y156R, N157P, F158N, P160T,S190E, S191A, K193E, K194R, N197E, W203R, G216N, D220R, K221E, K229Q,R231E, G232E, E235R, K238E, E240T, N287Q, D290R, E297H, K302Q, R303E,E307Q, D313P, K318D, K321D, K324P, R325Q, L328E, E343D, E345P, K348E,M349E, N352R, K353Q, I364E, K365R, Q369R, S403R, N407E 237 pA15880 6S4R, K5Q, D6R, Y7N, L8G, R9Q, K10R, K11W, S21E, E25R, E29R, E33Q, K34R,G35D, D36Q, Q49H, F50R, E59R, K62R, N63R, M66E, K67Q, K70R, K72E, E76P,D77R, K78R, E94R, S96E, T98E, N101E, K102E, K104R, D105E, K116E, P130S,K131E, V132T, P134D, E136R, K137E, E140R, E147R, I148E, R154T, K155T,Y156D, N157P, F158N, Q159R, P160T, V179R, S190E, S191Y, K193R, K194Q,N197Q, G216T, Y219R, D220R, K221R, K229Q, G232E, K238R, E240T, N287E,D290R, E297R, K302R, R303E, E307R, K321E, K324D, R325E, L328E, W331E,E343D, E345P, K348R, M349R, N352E, K353E, E356R, I364E, K365R, Q369R,D377H, E380D, K382R, K384R, K392E, K393Q, K396R, R397E, S403R, N407E 238pA15881 6 S4E, K5R, D6H, L8N, R9L, K10E, K11R, S21P, K32R, E33N, K34R,G35H, D36L, Y37P, Q49I, E59T, K62R, N63D, K67D, K70R, E71R, E75D, D77R,K78D, E94L, S96A, T98E, N101D, K102R, K104R, D105Q, K116R, D129Q, K131E,P134H, Y135W, E136D, K137E, R141L, E147R, I148V, T152V, R154Q, K155D,N157P, F158N, Q159L, P160L, V179I, S190E, S191A, K193R, K194R, P200L,G216R, K221R, E224M, K229R, G232E, K238R, E240V, D241P, E262Q, N287L,E297R, N300E, K302R, D313P, K318D, K321D, K324D, E327Y, L328W, W331L,E343D, E345P, K348R, M349T, N352E, K353R, E356R, G361P, D363P, I364L,K365E, F366H, Y368R, Q369D, F376E, E380D, K382R, K384E, S403A, N407D,L408T 239 pA15882 6 L3E, S4R, K5R, D6E, L8G, R9Q, K10R, K11E, S21R,K32R, E33T, K34R, G35H, Y37R, Q49H, F50R, N63R, F64E, K67R, K70R, E75D,D77R, K78R, E94R, S96R, T98E, K102E, K104R, D105Q, K116R, D129E, Y135W,K137E, R141Q, E144Q, I148E, E151R, T152R, A153R, R154T, K155E, V161E,S187R, S190E, S191E, K194R, N197P, W203R, Y219R, I292Q, S299Q, K318D,K321D, D322T, K324D, L326H, E327R, L328E, M349R, N352E, K353E, E356R,I364P, K365E, Y368R, Q369R, F376E, E380N, K382R, K384R, S403R, N407E,L408R 240 pA15883 6 S4R, K5H, D6Q, L8G, R9Q, K10E, K11E, S21E, E25R,E29R, K32Q, E33R, G35H, D36E, Y37R, Q49H, F50R, E59R, K62R, N63E, F64E,K67R, K70R, K72E, E75D, D77R, K78Q, E94R, S96R, T98E, K102E, K104R,D105Q, K116R, D129E, K137E, E140R, R141Q, I148E, E151R, T152R, V161E,S187R, S190E, S191E, K194R, N197P, W203R, Y219R, K229Q, G232E, K238R,E262Q, N287E, D290R, I292Q, K302R, R303E, E307R, K321D, D322T, K324D,L326H, E327R, L328E, W331E, E343D, E345P, K348R, M349E, N352R, K365R,Q369R, D373R, F376E, K382R, K384Q, K392H, K393R, K396E, R397E, E400Q,S403R, N407E, L408R 241 pA15884 6 L3E, S4E, K5H, D6R, Y7H, L8G, R9Q,K10E, K11F, S21E, K34Q, D36T, Q49H, F50R, K62R, N63R, M66E, K67E, K70R,E71R, E75D, D77R, K78M, E94Q, S96R, K102E, K104R, D105E, K116T, K131E,Y135D, K137E, E140R, I148E, E151R, T152H, A153R, R154Q, K155Q, N157P,F158T, Q159E, S187R, S190E, S191A, K193Q, K194R, N197P, P200E, W203R,S218T, Y219R, K221R, E224Q, K229R, G232E, K238R, E240T, E262R, N287E,D290R, E297R, S299Q, N300D, K302R, K318D, K321E, D322T, R325Q, L328E,W331E, E343D, E345P, K348R, M349R, K353E, E356R, I364E, K365R, F376Q,E380D, K382R, K384T, N407H, L408R 242 pA15885 6 S4R, D6Q, Y7N, L8N, R9E,K11R, S21P, K32R, K34R, G35N, N63R, K67E, K70R, E71S, E75D, D77R, K78R,E94L, T98E, N101R, K102R, K104E, D105E, K116R, K131E, V132T, Y135W,E144R, E151R, R154P, K155E, Y156V, N157P, F158N, Q159E, P160M, S191T,K193E, K194R, N197E, W203R, E240T, N287Q, D290R, E297H, N300D, K302R,K321D, K324D, R325Q, M349R, N352E, K353E, E356R, I364E, K365R, E380N,K382R, S403A, N407Q 243 pA15886 6 S4E, D6Q, Y7N, L8N, R9Q, K10D, K11R,S21P, K32R, K34R, G35D, D36Q, N63R, K67E, K70R, E71Q, K78M, S96E, N101E,K104R, D105Q, K116R, D129Q, K131E, V132T, P134D, Y135W, E136R, K137E,E140R, R141L, E151R, R154P, K155E Y156V, N157P, F158N, Q159E, P160M,S190E, S191T, K194R, N197S, W203A, G216N, D220R, K221E, K229R, G232E,R258E, E262R, N287Q, D290R, E297H, K302E, K321D, K324D, R325Q, L328E,E343D, K348R, M349R, N352E, K353E, E356R, I364E, K365R, E380N, K382R,K384E, S403A, N407E 244 pA15887 6 S4R, K5H, D6E, L8G, R9Q, K10R, K11E,S21R, K32Q, E33T, K34R, G35H, D36E, Y37R, Q49H, F50R, N63R, F64E, K67R,K70R, E75D, E94R, T98E, N101E, K102E, D105E, K116R, D129E, K131E, A153R,R154T, K155Q, V161E, S190E, S191Q, K193Q, K194R, Y195H, N197P, P200E,W203R, Y219R, K229Q, G232E, K238R, E240T, N287E, D290R, E297R, S299Q,N300D, K302R, K318N, K321D, D322T, K324D, R325E, L326H, E327R, L328R,W331E, E343N, E345P, K348E, M349E, N352R, K353R, I364P, K365E, Y368R,Q369R, F376E, K382R, K384Q, S403R, N407E, L408R 245 pA15888 6 S4E, K5H,D6H, L8G, K10P, K11Q, S21E, E25R, E29R, K32Q, E33R, K34Q, G35H, D36H,Y37P, Q49H, F50R, K62R, N63R, M66E, K67Q, K70R, K72E, D77R, K78M, E94R,S96E, T98E, K102E, K104R, D105E, K116W, P130S, K131E, V132T, P134D,E136R, K137R, E147R, I148E, K155G, N157P, V179R, S190E, S191Y, K193R,K194Q, P200R, G216R, Y219R, K221R, K229Q, G232E, K238R, E240H, N287E,D290R, E297R, N300D, K302R, K318D, K321E, D322T, K324E, R325Q, L328R,E343D, E345P, K348R, M349E, N352R, K353R, G361P, K365R, Q369R, F376E,E380D, K382R, K384Q, N385P, K392L, K393H, K396E, R397E, E400R, L408D 246pA15889 6 S4R, K5H, D6H, L8N, R9E, K10P, K11Q, S21P, K32Q, E33H, K34R,G35H, D36E, Y37P, F50H, K62R, N63R, K67E, K70R, E71R, E75D, D77R, K78L,E94Q, S96E, T98E, K102Q, K104Q, D105E, K116L, D129Q, K131E, K137E,R141H, E147R, I148V, R154Q, K155D, N157P, P160L, S190E, S191H, K193R,K194Q, N197R, G216R, K221R, K229Q, G232E, K238R, D241P, N287Q, K302R,R303Q, D313P, K318D, K321D, K324D, L328E, E343D, K348R, M349R, N352E,K353R, E356R, G361P, K365R, Q369R, F376E, E380D, K382R, K384E, N385P,S403R, N407D, L408T 247 pA15890 6 L3E, S4E, D6R, Y7H, L8G, K10E, K11R,S21E, K32I, K34M, D36Y, Q49W, F50S, M56W, K62R, N63R, M66E, K67E, K70R,E71R, E75D, E76P, K78M, E94L, S96E, T98R, N101E, K102Y, K104R, D105E,K116L, D129R, K131D, V132E, Y135R, K137E, E140R, R141W, E144D, E151R,T152Y, A153R, R154Q, K155T, Y156F, N157R, F158T, Q159E, S190E, S191Y,K193Q, K194R, N197P, W203R, S218T, Y219R, D220R, K221R, E224Q, K229R,G232E, K238R, E240T, E262R, N287E, D290R, E297R, S299Q, K302R, R303E,E307R, K318D, K321E, D322T, K324D, L328E, W331E, E343D, E345P, K348R,M349E, N352R, K353R, I364P, K365R, Q369R, F376E, E380R, K382Q, K384T,S403A, N407W, L408D 248 pA15891 6 S4R, K5H, D6Q, Y7N, L8N, R9E, K10P,K11V, S21P, E29R, F30A, K32Q, E33Q, K34T, G35N, D36T, F50H, K62R, N63E,K67R, K70R, E71S, K72T, E75D, D77R, K78R, E94Q, S96E, T98E, N101E,K102Q, K104R, D105Q, K116L, K131E, V132T, P134D, E136R, K137R, E151R,A153S, R154P, K155P, Y156R, N157P, F158N, P160T, S190E, S191H, K193R,K194Q, N197S, W203H, G216N, D220R, K221T, K229Q, R231E, G232E, E235R,K238R, E240T, R258E, E262R, N287Q, D290R, E297H, N300D, K302R, D313P,K318D, K321D, K324P, R325Q, L328E, E343D, K348R, M349R, N352E, K353Q,E356R, I364E, K365R, E380N, K382R, K384S, K392L, K393A, K396E, R397H,E400R, S403W, N407E 249 pA15892 6 S4R, D6Q, Y7N, L8N, R9E, K10P, K11V,S21P, K32M, K34L, G35D, D36L, K62R, N63E, K67R, K70R, E71S, E75D, D77R,K78M, K104R, D105E, K116Q, K131E, V132T, Y135W, E151R, R154P, K155P,Y156V, N157P, F158N, Q159T, P160M, S191T, K193R, K194R, N197S, W203A,D204R, G216N, D220R, K221T, K238R, E240T, E297H, K318D, K321D, K324P,R325Q, L328E, E345P, K348R, M349E, N352R, K353L, I364E, K365R, K384Q,N407R 250 pA15893 6 L3E, S4R, D6Q, Y7N, L8G, R9E, K10R, K11H, S21E,K32I, E33T, K34R, G35H, D36E, Y37R, K62R, N63R, F64E, M66E, K67R, K70R,E75D, D77R, K78L, K104E, D105E, K116Y, D129L, K131E, P134D, Y135W,E136R, K137E, E144R, T152R, A153R, R154T, K155Q, Y156Q, F158T, Q159E,P160F, V179I, S190E, S191W, K193R, K194R, N197P, W203R, Y219R, K229A,K238R, N287E, D290R, I292E, E297R, S299Q, N300D, K302R, Y315W, K318N,K321D, D322T, K324D, L326H, E327R, L328E, W331E, E343D, E345P, K348R,M349E, N352E, K353R, E356R, K365R, F366R, Q369E, F376E, K382R, K384R,S403R, N407E, L408N 251 pA15894 6 S4R, D6Q, Y7N, L8N, R9E, K10P, K11V,S21P, K34L, G35N, K62R, N63R, K67E, K70R, E71S, E75D, D77R, K78M, E94L,S96E, T98Q, N101H, K102Y, K104E, D105Q, K116R, K131E, V132T, P134D,Y135W, E136R, K137R, E144R, E151R, R154P, K155P, Y156V, N157P, F158N,Q159E, P160M, S191T, K193R, K194R, N197S, W203A, D204R, G216N, K221R,E224Q, K229Q, G232E, K238R, E240T, R258E, E262R, E297H, K318D, M349R,N352E, K353L, E356R, I364E, K365R, E380N, K382N, K384S, E389D, K392R,K396A, R397L, S403A, N407Q 252 pA15895 6 S4R, D6Q, L8N, R9E, K10P, K11V,S21P, K32M, K34L, G35N, D36T, N63E, K67R, K70R, E71S, E75D, D77R, K78R,E94L, T98Q, N101E, K102Y, D105E, K116Q, K131E, V132T, R154P, K155P,Y156V, N157P, F158N, Q159T, P160M, S190E, K193R, N197S, K229Q, G232E,E240T, R258E, E262R, N287Q, D290R, E297H, N300D, K302R, D313P, K318D,K321D, K324P, R325Q, E343D, E345P, K348R, M349R, N352E, K353A, E356R,K384D, N385T, R397L 253 pA15896 6 L3E, S4R, K5R, D6Q, Y7N, L8G, R9Q,K11E, S21E, E25R, K34R, G35H, D36S, Y37R, Q49H, F50R, N63R, F64E, K67R,K70R, E75D, D77R, K78R, E94R, T98E, K102E, K104R, D105Q, K116R, E147R,I148E, T152R, V161E, S190E, S191Y, K194R, N197P, W203R, K238R, I292Q,K318D, E343N, E345P, K348R, M349E, N352R, I364P, K365E, Y368R, Q369R,F376E, E380D, K382R, K384D, E389R, K392E, N407H, L408R 254 pA15897 6S4E, K5R, D6E, L8G, S21E, E33T, K34R, Y37R, Q49H, F50R, E59R, K62R,N63E, F64E, K67R, K70R, K78R, E94R, S96R, T98E, N101E, K102E, D105E,D129E, K137E, R141Q, S190E, K193E, K194R, N197P, P200E, W203R, S218T,Y219R, K221R, E224Q, E240T, N287E, D290R, I292Q, N300D, K302R, K318D,K321D, D322T, R325E, L326H, L328R, W331E, E343D, E345P, K348R, M349E,N352R, K365R, F376E, K382R, S403R, N407E 255 pA15898 6 L3E, D6R, Y7H,L8G, K10E, K11H, S21E, Y22W, K32I, E33R, D36Y, M56W, K62R, N63R, M66E,K67E, E71R, E76P, D77R, K78M, S96E, N101Q, K104R, D105E, K131D, Y135D,K137E, E140R, E151R, T152Y, A153R, R154Q, K155Q, Y156F, S187R, S190E,S191A, K194R, N197P, W203L, Y219R, K229R, G232E, E262R, N287E, D290R,E297R, S299Q, N300D, K302R, K318D, K321E, D322T, K324D, R325Q, E327R,L328E, W331E, E343D, E345P, K348R, M349E, N352E, K353R, E356R, I364E,K365R, F366Y, F376Q, E380D, K382R, K384T, E389R, K392L, K396Q, R397L,S403A, N407Y, L408D 256 pA15899 6 S4R, D6R, Y7N, L8G, R9E, K10P, K11V,S21R, K32Q, E33Q, K34Y, G35N, D36R, Y37E, E59R, K62R, N63R, M66E, K67E,E71S, E76P, D77R, K78R, I80L, S96E, N101Q, K104R, D105E, K116R, K131E,V132T, P134D, Y135W, E136R, K137E, E140R, E151R, K155T, N157P, F158N,Q159E, P160F, V179R, S190E, S191W, K193R, K194R, N197Q, G216T, D220Q,K221R, K229N, G232E, K238R, E240T, N287E, D290R, E297H, K302R, R303E,E307R, D313P, K318D, K321R, K324E, R325E, E327R, L328R, W331E, E343N,E345P, K348E, M349R, N352Q, K353Q, E356R, I364E, K365R, Q369R, K384Q,K392R, K396R, R397I, S403A, N407E 257 pA15900 6 L3E, S4E, K5H, Y7H, L8N,R9Q, K10P, K11I, S21P, E25R, E33H, K34R, G35H, D36E, F50H, K62R, N63R,K67E, K70R, E71Q, K72Q, E75D, D77R, K78Q, E94R, S96E, T98E, N101E,K102E, K104R, D105Q, K116H, K137E, E140R, E150R, K155E, Y156E, N157E,Q159G, P160S, S190E, S191H, K193R, K194Q, N197Q, N201E, D204R, R205L,S218T, Y219R, K221Q, E224Q, K229R, G232E, E240N, N287R, K302R, R303E,E307R, K318D, K321D, K324D, E343D, K348E, M349R, N352R, G361D, I364E,K365R, Q369R, D373E, F376E, E380R, K382R, K384D, N385T, K393H, E400R,S403R, N407Q, L408R 258 pA15901 6 L3E, D6R, Y7H, L8N, R9E, K10P, K11R,S21P, Y22W, K32M, E33R, K34Q, K62R, N63Q, K67E, K70R, E71R, E75D, D77R,K78M, E94L, S96E, T98R, N101E, K102Y, K104R, D105Q, K116I, D129Q, K131D,V132R, Y135L, K137E, E140R, R141L, E151R, T152Y, R154Q, K155E, Y156F,N157D, F158R, Q159E, P160M, S190E, S191A, K193E, K194R, N197D, W203R,S218T, K221Q, K229R, R231E, G232E, E235R, K238Q, E240T, N287L, D290E,S299Q, N300D, K302R, D313P, K318D, K321D, K324D, E343D, K348R, M349R,N352E, K353L, E356R, I364E, K365R, F366Y, F376E, E380R, K382R, K384E,N385P, E389R, K392H, K396I, R397L, S403A, N407L, L408T 259 pA15902 6S4Q, K5Q, D6R, Y7N, L8G, R9Q, K11W, S21E, E25R, K32Q, K34S, G35N, D36T,N63R, K67Q, K70R, K72H, E75D, D77R, K78R, E94R, S96E, T98E, K102R,K104R, D105E, K116R, P134D, E136R, K137E, E140R, E147R, I148E, T152Q,A153S, R154S, K155T, Y156D, N157P, F158N, Q159E, P160T, V179R, S190E,S191E, K193R, K194R, Y195Q, N197Q, W203H, G216T, Y219R, D220R, K221R,K229Q, G232E, N287E, D290R, K302R, R303E, E307R, K321E, K324E, R325Q,E327R, L328E, W331E, I364E, K365R, Q369R, K384Q, K392R, K393R, R397E,S403R, N407E 260 pA15903 6 L3E, S4E, K5H, Y7H, R9T, K11I, S21P, K32Q,K34Q, F50H, N63R, K67E, K70R, E71R, E75D, D77R, K78M, S96E, T98E, N101E,K102Q, D105Q, K116Y, D129Q, 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K348E, M349R, N352R,K353Q, D377R, E380N, K382E, K384D, N385T, K392L, K393A, K396E, N407Q 266pA15909 6 L3E, S4E, K5H, R9Q, K11F, S21E, K34Q, D36T, Y37E, Q49H, F50R,K62R, N63R, M66E, K67E, K70R, E71R, E75D, I80R, E94Q, S96E, N101E,K102E, K104R, D105E, K116T, K131D, V132E, Y135R, K137E, E140R, I148D,E151R, T152H, R154Q, N157P, F158E, Q159E, S190E, S191R, K194E, N197P,W203R, Y219R, K229R, G232E, E297R, S299Q, K318D, K321E, D322T, K324D,R325Q, E327R, L328E, E343Q, E345D, K348R, M349E, N352R, K353R, N407H,L408R 267 pA15910 6 S4R, K5H, D6E, L8G, R9Q, K10P, K11E, S21E, E25R,E33R, G35H, D36E, Y37R, N63R, F64E, K67R, K70R, K72E, E75D, D77R, K78Q,S96R, K116R, D129E, P134D, E136R, K137E, R141Q, A153R, R154T, K155E,V161E, S190E, S191H, K193E, K194R, Y195H, N197P, P200E, W203R, S218T,Y219R, K221R, E224Q, K229Q, G232E, E240T, I292Q, K318N, K321D, D322T,L326H, L328E, K365R, Q369R, F376E, K382R, K384Q, E389R, K392H, K393H,K396E, E400R, S403R, N407E, L408R 268 pA15911 6 S4M, K5V, D6Q, Y7N, L8N,R9I, K10P, K11I, S21P, K32M, G35N, D36S, N63R, K67E, K70R, E71S, K78M,S96A, N101S, K104Q, D105E, K116L, D129Q, K131E, V132T, P134D, Y135W,E136R, K137E, E140I, R141L, E144I, I148V, E151R, T152V, R154P, K155P,Y156V, N157P, F158N, Q159L, P160M, S190E, S191W, K193Y, K194R, N197S,W203A, K238S, E262L, N287L, D290R, E297H, N300D, K302L, D313P, K318D,K321R, K324P, R325A, L328D, W331L, M349R, N352E, K353L, E356R, N407L 269pA15912 6 D6Q, L8N, S21P, K34L, G35D, D36Q, E75D, D77R, K78R, S96E,N101E, K104R, D105Q, K116R, D129Q, P134D, Y135W, E136R, K137E, E140R,R141L, E151R, R154P, K155E, Y156V, N157P, F158N, Q159E, P160M, S190E,S191T, K194R, N197S, W203A, K229R, R231E, G232E, E235R, K238E, D313P,K318D, K321D, K324P, R325Q, E343D, E345P, K348E, M349R, N352R, I364E,K365R, K384E, K392R 270 pA15913 6 L8N, R9E, K10P, S21P, E33H, K34I,G35H, D36E, Y37P, K62R, N63R, K70R, E75D, D77R, K78L, K104Q, D105E,K116R, E147R, R154Q, K155D, Y156F, N157P, Q159L, S190E, K193R, N197R,G216R, D220R, K221E, E240M, N287L, D290E, K302R, R303E, E307R, K324D,R325E, L328E, E343D, K348E, M349R, N352R, G361P, K365R, Q369R, F376E,E380D, K382R, K384E, N385P, S403A, N407F, L408T 271 pA15914 6 S4R, Y7N,L8N, R9E, K10P, K11V, S21P, K34L, G35S, Q49F, F50Q, N63E, K67R, K70R,E71S, K78M, E94L, S96E, T98Q, N101E, K102Y, D105Q, D129Q, K131E, V132T,P134D, E136R, K137E, E140R, R141L, A153S, R154P, K155P, Y156Q, N157P,F158N, S190E, K193R, N197S, K229Q, G232E, E240T, N287Q, D290R, K302Q,K321D, K324P, R325Q, E343D, E345P, K348E, M349R, N352R, I364P, K365R,F366Y, Q369R, Y375A, E380N, K382N, S403A, N407Q 272 pA15915 6 S4R, D6Q,Y7N, L8N, R9E, K10P, K11V, S21P, K32M, E33R, K34T, G35N, N63E, K67R,K70R, E71S, E75D, D77R, K78M, S96E, K116Q, D129Q, P134D, E136R, K137E,E140R, R141L, R154P, K155P, Y156V, N157P, F158N, Q159T, P160I, S190E,K193R, K194E, N197E, K229Q, G232E, E240T, N287Q, D290R, N300D, K302R,D313P, K318D, K321D, K324P, R325Q, K396M, R397L, S403L, N407Q 273pA15916 6 S4E, D6H, L8G, K10E, K11Q, S21E, Y22W, K32M, E33Q, K34L, G35H,D36N, Y37P, Q49W, F50S, M56W, K62R, N63R, M66E, K67E, K70R, E71R, E75D,D77R, K78L, E94L, S96E, T98Q, N101Q, K102Y, K104Q, D105E, K116E, K131E,V132T, P134D, Y135W, E136R, K137R, E147R, R154S, K155G, Y156F, N157P,Q159R, V161I, V179R, S190E, S191Y, K193R, K194Q, P200R, G216R, Y219R,K221R, K229Q, G232E, K238R, E240Y, E262Q, N287L, D290E, E297R, N300D,K302R, K318D, K321E, D322T, K324D, R325Q, E327R, E343D, E345P, K348R,M349E, N352R, K353R, G361P, I364P, K365R, Q369R, D373R, F376E, E380D,K382R, K384Q, N385P, K392R, K396R, R397L, L408D 274 pA15917 6 S4R, K5H,D6Q, L8N, R9E, K11V, S21P, F30H, K32Q, K34H, G35N, F50H, N63R, K67E,K70R, E71S, E75D, D77R, K78R, E94Q, S96E, T98E, N101E, K102Q, D105E,K116R, P134D, E136R, K137R, A153S, R154P, K155P, Y156Q, N157P, F158N,Q159E, P160T, S190E, K193R, N197S, W203H, K229Q, G232E, E240T, N287Q,D290Q, K302R, R303E, E307R, K321D, K324P, R325Q, E343D, K348E, M349R,N352R, K384Q, N407Q 275 pA15918 6 S4R, D6Q, Y7N, L8N, R9E, K11V, S21P,D36Y, N63E, K67R, K70R, E71S, E94L, T98Q, N101E, K102Y, D105E, K116R,K131E, V132T, A153S, R154P, K155P, Y156V, N157P, F158N, Q159E, P160M,S190E, S191T, K193R, K194Q, N197E, W203Y, K229Q, G232E, E240T, N287Q,D290R, K302Q, K318D, K321D, K324P, R325Q, I364E, K365R, K384Q, N407E 276pA15919 6 S4D, K5H, D6H, L8G, K10P, K11F, F30A, K32M, E33N, K34Q, G35H,D36Y, Y37P, Q49H, F50R, M56W, K62R, N63R, M66E, E75S, E76L, D77S, K78M,E94Y, T98W, K102E, K104R, D105E, P130S, K131E, V132T, Y135W, E136R,R141W, E147R, I148E, T152Q, K155G, Y156F, N157P, V179R, S190E, S191E,K193R, K194E, N197R, P200L, G216R, Y219R, K221R, D228S, K229Q, R231E,G232P, E235R, K238W, E240Y, E262L, N287L, E297R, N300E, K302P, D313P,K318D, K321E, D322T, K324D, R325Q, E327R, L328W, W331L, M349R, N352E,K353Q, E356R, G361P, D363P, I364P, K365E, Y368R, Q369R, Y375W, F376E,E380D, K382R, K384R, N385P, S403W, N407L, L408D 277 pA15920 6 L3E, S4R,K5H, D6E, L8G, R9Q, K10A, K11F, S21E, K32I, E33T, K34R, G35H, D36E,Y37R, Q49H, F50R, E59S, K62R, N63D, F64E, M66F, K67R, K70R, E75D, D77R,K78L, E94Y, S96R, T98W, K102E, K104R, D105Q, K116Y, D129E, K131E, Y135W,E136P, K137E, E144D, I148E, E151R, T152R, A153R, R154T, K155Q, Y156F,F158T, Q159E, V161W, V179I, S187R, S190E, S191W, K193R, K194R, N197P,P200M, W203Y, S218T, Y219R, K221R, E224Y, K229A, G232P, E235W, K238S,E240T, N287L, E297R, S299Q, N300E, K302L, D313P, Y315W, K318D, K321D,D322T, K324W, R325W, L326H, L328W, W331F, E343D, E345P, K348R, M349E,N352E, K353R, E356R, D363H, I364P, K365E, Y368R, Q369R, F376E, E380N,K382R, K384R, S403Y, N407F, L408W 278 pA15921 6 S4R, K5F, D6H, L8N, R9E,K10P, K11I, S21E, K32M, E33H, K34I, G35H, D36Y, Y37P, E59T, K62R, N63D,K70R, E75D, D77R, K78L, E94L, S96A, T98Q, N101T, K102Y, D105E, K116L,K131E, P134H, Y135W, K137E, P160L, V179I, S190Y, S191Y, K194E, N197R,G216R, K221V, D228S, K229Q, R231E, G232E, E262L, E297R, N300Q, D313P,K318D, K321D, K324D, E327I, L328W, E343D, E345P, K348Y, M349Y, N352L,K353L, E356W, G361P, D363P, I364L, K365E, F366H, Y368R, Q369D, F376E,E380D, K382R, K384S, S403A, N407W, L408T 279 pA15922 6 L3E, S4L, K5H,Y7N, L8G, R9Q, K10R, K11F, S21E, E33N, K34R, G35H, D36W, Y37R, N63R,F64E, K67R, K70R, D77N, K78M, K116Y, D129E, K131E, Y135W, E136P, A153R,R154T, K155Q, Y156Q, F158T, Q159E, P160F, V161W, V179I, S190E, S191W,K194R, N197P, W203L, S218T, Y219R, K221T, E224Y, K229A, G232P, N287L,E297R, S299Q, N300E, K302L, K321S, D322T, R325S, L326H, L328W, W331F,E343D, E345P, K348R, M349E, N352R, K353R, D363H, I364P, K365S, Y368R,Q369D, F376E, K382R, S403W, N407Y, L408W 280 pA15923 6 L3E, S4E, L8G,R9Q, K10R, K11R, S21E, K34R, D36E, F50H, M56W, K62R, N63Q, M66E, K67E,K70R, E71R, E75D, E76P, E94L, S96E, T98E, N101R, K102E, D105E, K131S,E136Q, K137E, E140R, E150A, K155D, Y156Q, N157E, Q159G, P160R, S190E,S191Y, K193R, K194R, N197S, N201R, R205Q, Y219R, K229Q, G232E, N287R,N300D, K302R, K321E, L326H, E343Y, E345P, K348R, M349E, N352R, I364E,K365R, Y368R, Q369R, F376E, E380N, K382R, S403W, L408R 281 pA15924 6S4E, K5H, D6H, L8G, R9Q, K11Q, S21Q, K34Q, G35H, D36H, Y37P, Q49H, F50R,K62R, N63R, M66E, K67E, K70R, E71R, E75D, D77R, K78L, E94R, T98E, N101E,K102E, K104R, P130S, K131E, V132T, E136R, E147R, I148E, V179R, S190E,S191Y, K194E, N197R, W203R, Y219R, K229Q, G232E, K238R, K321E, D322T,K324D, R325Q, E327R, L328E, E343Q, E345P, K348Q, M349E, N352R, G361P,I364E, K365E, Y368R, Q369R, K384R, N385P 282 pA15925 6 S4L, K5H, D6Q,L8G, R9Q, K10R, K11F, S21E, Y22W, E25R, E29Q, K32I, E33T, K34R, G35H,D36H, Y37R, Q49H, F50R, N63R, F64E, K67R, K70R, K72E, D77N, K78M, E94Y,S96R, T98W, N101E, K102E, D105E, K116Y, D129L, K131E, P134H, Y135W,E136S, K137E, R141Q, A153R, R154T, K155Q, Y156Q, F158T, Q159E, P160F,V161Y, V179I, S190E, S191Q, K193R, K194R, Y195H, N197P, P200M, W203Y,Y219R, K229A, G232P, E240T, I292Q, K321D, D322T, R325D, L326H, L328W,E343D, E345P, K348R, M349E, N352R, D363H, I364P, K365S, Y368R, Q369D,D373R, F376E, K382R, K384R, K392F, K393R, K396R, R397E, E400Q, S403Y,N407F, L408W 283 pA15926 6 S4R, D6H, L8N, R9E, K10P, K11Q, S21P, E33H,K34R, D36E, K155D, N157P, G216R, K221R, K229Q, G232E, K238R, D241P,N287Q, K302R, K321D, K324D, E343D, K348E, M349R, N352R, G361P, K365R,Q369R, K384E, N385P, S403R, N407D, L408T 284 pA15927 6 S4D, K5A, D6H,L8G, K10P, K11F, S21E, Y22W, E25R, E29R, E33R, K34L, G35H, D36Y, Y37P,Q49H, F50R, N63R, K67R, K70R, K72L, D77N, K78M, E94Y, T98W, N101Q,K102E, D105E, K116W, P130S, K131E, V132T, K155G, Y156F, Q159W, V161I,G216R, Y219R, K221R, K238S, E262L, N287E, D290R, E297R, N300R, K302R,R303D, K318D, K321E, D322T, K324D, R325Q, E327R, L328Y, W331E, E343N,E345P, K348E, M349E, N352R, K353R, G361P, D363P, I364P, K365E, Y368R,Q369R, F376E, E380D, K382R, K384Q, N385P, K392F, K393H, K396E, R397L,E400R, S403A, N407L, L408D 285 pA15928 6 S4R, K5H, D6Q, Y7N, L8N, R9E,K10P, K11V, S21P, E25R, K34T, G35N, N63R, K67E, K70R, E71Q, K72T, K78R,N101E, K104R, D105Q, D129Q, K131E, V132T, R141Q, E151R, A153S, R154P,K155P, Y156Q, N157P, F158N, K229Q, G232E, R258E, E262R, N287Q, D290R,E297H, K302Q, R303E, E307Q, D313P, K318D, K321D, K324P, R325Q, E343D,K348R, M349R, N352E, K353Q, E356R, I364E, K365R, E380N, K382N, S403R,N407Q 286 pA15929 6 L3E, S4Q, K5H, D6R, L8N, K11Y, K32Q, D36T, E75D,D77R, K78M, K116Y, K131D, V132R, A153Q, R154Q, K155E, Y156T, N157D,F158R, K193E, N197D, W203R, K229R, G232E, E240T, S299Q, D313P, K318D,M349R, N352E, K353R, E356R, I364E, K365R, Q369R, F376E, E380R, K382R,K384E, N385P 287 pA15930 6 S4L, K5V, D6Q, Y7W, L8N, R9I, K11I, S21P,Y22W, E25I, E29L, F30T, K32M, K34F, G35N, D36Y, E59S, K62R, N63D, M66Y,K67E, K70L, E71Q, K72L, E76L, K78M, E94L, S96A, T98R, N101T, K102Y,K104R, D105Q, K116L, D129Q, K131E, V132T, P134D, Y135W, E136R, K137Q,E140I, R141Y, I148V, E151R, T152V, R154P, K155P, Y156V, N157P, F158N,Q159L, P160M, S190E, S191T, K193L, K194R, N197S, W203A, D204S, D228S,K229Q, R231L, G232P, E240T, E297H, D313P, K318D, K321R, K324P, R325A,L328D, M349R, N352E, K353L, E356R, D377A, K384S, E389D, K392L, K393W,R397L, E400L, S403W, N407I 288 pA15931 6 S4R, D6H, L8G, R9E, K10P, K11Q,S21E, Y37P, K116E, K155G, Y156F, N157P, Q159R, V161I, V179R, G216R,Y219R, K221R, V222I, K229Q, G232E, N287E, D290R, N300D, K302R, K321E,R325Q, W331E, E343D, E345P, K348R, M349E, N352R, G361P, F376E, E380D,K382R, K384R, N385P, L408N 289 pA15932 6 S4M, K5V, D6Q, Y7N, L8N, R9L,K10P, K11Y, S21P, F30A, K34Y, G35N, D36F, N63E, K67R, K70R, E71S, K78M,S96A, D129Q, K131E, V132T, P134D, E136R, K137I, E140I, R141Y, R154P,K155P, Y156V, N157P, F158N, P160M, K238S, E262L, N287L, D290R, E297H,N300D, K302L, K321R, K324P, R325A, W331F, E343M, E345P, K348W, M349T,N352M, K353L, I364E, K365R, Q369L, S403W, N407I 290 pA15933 6 S4L, K5V,D6Q, Y7W, L8N, R9I, K10P, K11I, F30A, K32M, K34T, G35D, D36Q, K70R,E71S, K78M, S96A, D129Q, K131E, V132T, P134D, E136R, K137Q, E140I,R141Y, R154P, K155P, Y156V, N157P, F158N, P160M, K193L, N197S, W203A,D204N, D228S, K229Q, R231E, G232E, E235R, K238S, E240T, E262L, N287L,D290R, N300D, K302L, D313P, K318D, K321R, K324P, R325A, W331L, K384L,S403W, N407L 291 pA15934 6 S4R, D6Q, L8G, R9H, K11E, S21R, K34L, G35H,D36S, Y37R, N63R, F64L, K67E, K70R, E71R, E75D, D77R, K78L, N101Q,K104R, D105E, K116Y, D129E, K131E, E144Q, E151R, T152Y, A153R, F158T,S190E, K193R, K194R, N197P, W203R, Y219R, E240T, N287E, D290R, N300D,K302R, K321D, D322T, R325E, L326H, L328R, W331E, E343Y, E345P, K348E,M349R, N352R, D373N, F376E, K382R, K384Q, R397S, N407H, L408R 292pA15935 6 S4E, K5L, D6L, Y7W, L8N, R9Q, K10P, K11V, F30T, D36W, Q49F,F50Q, E59S, K62R, N63D, M66Y, K67Q, E76L, E94L, S96A, T98Q, N101E,K102Y, D105I, K116L, P125A, D129Q, K131Y, V132Y, P134D, E136R, K137Q,E140I, R141Y, R154P, K155P, Y156V, N157P, F158N, Q159L, P160M, S190E,S191T, K194R, N197S, W203F, E262L, N287L, D290R, N300D, K302L, D313P,K318D, W331L, I364E, K365R, F366Y, Q369L, D377A, E380R, K382E, K393W,K396I, R397L, E400Y, S403W, N4071 293 pA15936 6 S4M, K5V, D6Q, L8N, R9L,K10P, K11Y, S21P, F30A, K34Y, G35N, D36F, K70D, E71S, E76R, K78M, Y135W,R154P, K155P, Y156V, N157P, F158N, P160M, S190E, S191T, K193Y, K194R,N197S, W203A, E262L, N287L, D290R, N300D, K302L, W331L, E343D, K348A,M349R, E380L, K382R, S403W, N4071 294 pA15937 6 S4M, K5L, D6Q, R9I,K10N, K11I, S21P, F30A, K32Q, K34Y, G35D, D36L, E59S, K62R, N63D, K67E,K70D, E71R, E76R, D77N, K78R, S96A, N101A, K104E, D105E, K116T, K131E,V132T, Y135L, K137V, E144R, I148V, E151R, K155E, F158N, P160E, S190D,S191T, K193R, K194R, N197E, W203Y, D204N, E240T, E262L, N287F, D290Q,K302L, D313P, K318D, K321D, K324E, W331L, E343N, E345P, K348E, M349T,N352R, I364P, K365R, E380R, K382L, S403F, N4071 295 pA15938 6 S4E, D6H,L8G, K11H, S21E, K32M, E33Q, G35D, D36E, Y37P, Q49F, F50S, M56W, K62R,N63R, M66E, K67E, K70R, E71R, E75D, E76P, I80Q, E94Y, S96E, T98Q, N101Q,K102W, K104R, D105E, K116E, D129W, K137V, R141W, E147R, Y156F, Q159R,V161I, V179R, S191L, G216R, Y219R, K221R, K229Q, G232E, K238R, K321E,D322T, K324D, R325Q, E327R, E343D, E345P, K348R, M349E, N352R, G361P,K365R, Q369R, F376E, E380R, K382Q, K384R, N385P, N407H, L408D 296pA15939 6 S4M, K5L, D6Q, R9I, K10N, K11I, S21P, F30H, K32Q, K34T, G35N,E59S, K62R, N63D, K67S, D77N, K78R, S96A, N101E, K104R, D105Q, K116T,K131E, V132T, Y135L, K137V, I148V, E151R, K155E, F158N, P160E, S191T,K193R, K194D, N197S, W203Y, D204N, G216R, K221Q, D228S, K229Q, R231E,G232E, E235R, K238S, E240T, E262L, D290R, E297L, K302L, D313P, K318D,K321D, K324E, W331L, E345P, K348E, M349E, N352R, K353Q, I364P, K365R,F366Y, K384Q, S403T, N407Q 297 pA15940 6 S4M, K5V, D6Q, L8N, R9L, K10P,K11Y, S21P, Y22W, F30T, E33Q, K34Y, G35N, D36F, E59S, K62R, N63D, K67E,K70R, E71Q, E75D, D77R, K78M, E94L, T98Q, N101E, K102Y, D105E, K116L,Y135W, Q159E, P160M, S191T, K193L, K194A, N197S, W203A, D204R, E240T,E262L, N287L, D290R, N300D, K302L, D313P, K318D, K321R, K324P, R325A,W331L, I364E, K365R, F366Y, Q369L, D377A, K384S, E389D, K392L, K393W,K396I, R397L, E400Y, S403W, N4071 298 pA15941 6 S4M, K5H, D6E, L8G,K11F, E33N, K34R, G35H, D36H, Y37R, E75D, D77R, K78L, S96R, K104R,D105E, K116Y, D129E, P134H, Y135W, E136S, K137R, R141D, E144D, I148E,E151R, T152R, Y156E, F158T, P160F, V161E, V179I, S187Q, S190E, S191Q,K193I, K194R, Y195H, N197P, P200L, W203L, S218T, Y219R, K221T, E224Y,K229A, G232P, E240T, I292Q, K321S, D322T, R325S, L326H, L328W, E343Y,E345P, K348E, M349W, N352R, D363H, I364P, K365S, Y368R, Q369D, F376E,K382R, S403Y, N407F 299 pA15942 6 L3E, S4R, K5H, Y7N, R9Q, K11F, S21E,Y22W, E25R, E29R, E33T, K34R, D36E, Y37R, Q49H, F50R, E59S, K62R, N63D,F64E, M66Y, K67R, K70R, K72E, K78R, E94Y, T98W, N101E, K102E, D105E,K116Y, D129E, K131E, Y135W, E136P, A153R, R154T, K155Q, Y156F, F158T,Q159E, P160F, V161W, V179I, S190E, S191W, K194R, N197P, W203L, S218T,Y219R, K221T, E224Y, K229A, G232P, N287L, D290F, I292Q, K302L, R303E,E307R, K321S, D322T, R325S, L326H, L328W, W331F, D363H, I364P, K365S,Y368L, K384R, K392H, K396M, S403Y, N407F, L408W 300 pA15943 6 S4D, K5Q,D6R, Y7N, L8G, R9Q, K10P, K11W, S21E, E25R, K32Q, G35D, D36Q, K62A,N63R, M66Y, K67E, K70R, K72H, E75R, K78R, N101Q, K104R, D105E, K116Y,Y135W, E136P, I148V, E151R, T152V, R154Q, K155W, N157P, F158N, Q159L,P160F, V179R, S190E, S191W, K193R, K194R, N197E, P200L, D228S, K229Q,R231F, G232P, E240T, D241R, E262L, E343D, E345P, K348R, M349R, D363H,I364E, K365S, Q369R, E380N, K382N, N407E 301 pA15944 6 S4E, K5H, D6H,L8G, K10P, K11F, S21E, K34L, G35H, D36Y, Y37P, M56W, E59S, K62R, N63D,M66F, K67E, E71R, E75D, E76L, D77R, K78L, V179R, G216R, Y219R, K221R,D228S, K229Q, G232P, E262L, N287L, N300D, K302P, K321E, D322T, K324D,R325Q, E327R, L328W, W331L, E343D, E345P, K348R, M349E, N352R, G361P,D363P, K365D, Y368R, Q369R, K384R, N385P, L408D 302 pA15945 6 S4R, K5F,D6H, L8N, R9E, K10P, K11I, S21P, F30A, K32M, E33H, K34V, G35H, D36E,Y37P, Q49W, F50Q, E59T, K62R, N63D, K67T, K70R, E75D, D77R, K78L, E94L,S96A, T98Q, N101T, K102Y, K104Q, D105I, K116L, D129W, P130S, K131E,Y135W, K137L, R141L, E147R, I148V, T152V, A153G, R154Q, K155D, Y156M,N157P, Q159L, P160M, V179I, S190Y, S191Y, K193F, K194L, P200L, W203R,G216R, K221V, E224M, D228S, K229Q, R231E, G232E, E235R, K238S, E240Y,D241P, E262L, N287L, E297R, N300Q, K302I, D313P, K318D, K321D, K324D,E327Y, L328W, W331L, E343L, E345P, K348W, M349Y, N352L, K353L, E356W,G361P, I364L, K365R, Y368L, Q369R, F376E, E380D, K382R, K384S, S403W 303pA15946 6 S4P, K5A, D6H, K10E, K11H, S21R, K32Q, E33N, K34M, G35H, D36E,Y37P, M56W, E59S, K62R, N63D, M66Y, E75R, E76L, K78L, E94L, S96E, T98I,N101Q, K102Y, K104R, D105E, K131E, V132T, P134D, Y135L, E136T, K137R,E147R, I148E, V179I, S190D, S191R, K193R, K194E, G216R, K221Q, K229Q,G232P, E262Q, N287R, K302R, R303E, E307R, K321R, D322T, K324E, R325D,E327R, L328Y, E345P, K348Q, M349E, N352R, K353R, G361P, D363P, I364P,K365N, F376E, E380R, K382L, K384R, N385P, S403A, N407H, L408D 304pA15947 6 S4D, K5H, D6H, R9L, K10N, K11I, S21P, K32A, E33N, K34M, G35H,D36L, Y37P, E59S, K62R, N63D, K67D, K70D, E71R, E75D, E76R, D77N, K78L,E94L, S96A, T98Q, N101D, K102Y, K104R, D105Q, K116T, K131E, K137V,E147R, I148V, K155E, P160A, V179I, S190E, S191L, K193R, K194R, G216R,K221I, D228S, K229Q, R231E, G232E, E235R, K238S, D241N, E262L, N287H,D290E, K302I, R303E, E307R, D313P, K318D, K321D, K324E, L328Y, W331L,E345P, K348R, M349T, N352E, K353L, E356R, G361P, D363P, I364L, K365S,F376E, E380R, K382L, K384E, S403A 305 pA15948 6 S4D, K5A, D6H, K10D,K11I, S21P, E25R, E29I, K32I, E33R, K34T, E59S, K62R, N63D, K67S, K70D,K72L, E75D, E76R, D77N, K78L, E94L, S96A, T98R, N101E, K102Y, K104R,D105Q, R108I, K116T, K131E, V132R, Y135L, K137V, E140R, E151R, T152Y,R154Q, K155E, F158V, S187A, S190E, S191A, K193R, K194R, N197D, W203F,D220E, K221T, D228S, K229R, R231E, G232E, E235R, K238S, E240D, D241Q,R258L, E262R, N287H, D290E, S299A, K302I, R303E, E307R, D313P, K318D,K321D, K324E, L328Y, W331L, E345P, K348L, M349T, N352E, K353R, E356R,I364M, K365R, F366Y, F376E, D377R, E380R, K382E, K384T, N385P, K392L,K393T, K396M, R397L, S403A, L408D 306 pA15949 6 L3T, S4D, K5H, D6N,K10E, K11I, S21E, K32I, K34M, D36S, M56W, E59S, K62R, N63D, M66F, K67E,K70D, E71R, E76R, D77N, K78L, E94L, S96E, T98R, N101R, K102Y, K104E,D105E, K116T, K131E, Y135L, K137R, E144R, I148V, E151R, T152Y, A153R,R154Q, K155Q, F158V, S187A, S190E, S191A, K193R, K194R, N197S, P200M,W203Y, D220Q, K221T, E224Y, D228S, K229R, R231E, G232P, E235R, K238S,E240T, E262Q, N287E, D290R, K302I, R303E, E307R, D313P, K318D, K321R,D322T, K324E, R325D, L328Y, W331L, E343N, E345P, K348R, M349E, N352R,K353R, I364P, K365R, F366L, F376E, E380R, K382L, K384T, S403A, N407H,L408D 307 pA15950 6 S4M, K5L, D6Q, R9I, K10N, K11I, E25R, E29I, F30A,K32L, E33R, K34Y, G35D, D36L, E59S, K62A, N63E, M66Y, K67R, K70Q, K72I,D77N, K78R, S96A, K116T, K131E, V132T, Y135L, K137V, K155E, F158N,P160E, S191T, K193R, K194D, N197S, W203Y, D204N, G216R, K221Q, D228S,K229Q, R231E, G232E, E235R, K238S, E240T, E262L, N287F, D290Q, K302L,D313P, K318D, K321R, K324E, R325D, W331L, E345P, K348E, M349T, N352R,D377S, E380R, K382L, K384S, E389D, K392L, K393Y, K396M, R397L, S403F,N4071 308 pA15951 6 S4N, K5Q, D6Q, K10R, K11E, S21P, K32Q, K34Y, G35N,D36R, E59S, K62R, N63D, M66F, K67E, K70D, E71R, E76R, D77N, K78R, S96E,K104R, D105E, P130S, K131E, V132T, E136P, K137R, E144I, I148E, E151R,K155E, F158N, S190E, S191Y, K193R, K194R, N197L, G216R, K221Q, K229N,G232P, D241R, K321R, K324E, R325D, E327R, E343N, E345P, K348E, M349N,N352R, I364P, K365R, S403A, N407E 309 pA15952 6 S4L, K5L, D6Q, K10N,K11I, S21P, N63R, K67E, K70D, E71R, E76R, E94L, S96A, T98Q, N101E,K102Y, D105E, K116T, Y135L, K137V, K155E, F158N, P160E, S190D, S191T,K193R, K194R, N197S, W203Y, K238S, E262L, N287F, D290Q, K302L, D313P,K318D, W331L, I364P, K365R 310 pA15953 6 S4M, K5L, D6Q, R9I, K10N, K11I,S21P, E25R, K32L, K34T, G35N, E59S, K62R, N63E, K67R, K70Q, K72I, K78R,N101A, K104E, D105E, K131E, V132T, Y135L, E144R, I148V, E151R, K155E,F158N, P160E, S190D, S191T, K193R, K194R, N197S, W203Y, D204N, D228S,K229Q, R231E, G232E, E235R, K238S, E240T, E262L, E297L, D313P, K318D,K321D, K324E, M349I, N352E, K353L, E356R, I364P, K365R, D377R, E380N,K382D, K393Y, S403L, N407Q 311 pA15954 6 K5A, D6Q, S21P, K32Q, K34Y,G35N, D36T, N63R, K67E, K70D, E71R, E76R, D77N, K78R, N101A, K104E,D105E, Y135L, E144R, I148V, E151R, K155E, F158N, P160E, S190E, S191T,K193E, K194R, N197S, W203F, G216R, K221Q, K238S, E297L, K321D, K324E,M349I, N352E, K353L, E356R, I364P, K365R, K384Q 312 pA15955 6 L3T, S4D,K5H, K10R, K11V, S21E, E25R, E29R, K32R, E33D, K34D, G35D, D36T, F50H,E59S, K62R, N63D, M66Y, K67R, K70D, K72E, E75R, E76R, K78L, E94R, S96E,T98E, K102E, K104R, D105E, D129R, K131S, V132S, E136P, K137R, I148E,E151R, T152Y, R154E, K155Q, F158L, S190E, S191Y, K193R, K194E, N197R,P200E, D228S, K229Q, R231E, G232P, E235R, K238S, E240T, N287R, D290E,K302I, R303E, E307R, K318N, K321R, K324E, R325G, L328Y, W331L, D363P,K365N, D373E, F376E, D377N, E380R, K382L, K384Q, E389R, K392L, K393E,K396M, E400R, S403A, N407R 313 pA15956 6 S4M, K5L, D6Q, R9I, K10N, K11I,S21P, E29R, K32Q, E33R, K34T, G35N, D36L, N63R, K67E, K70D, E71R, K72T,E76R, D77N, K78R, E94L, S96A, T98Q, N101E, K102Y, D105E, K131E, V132T,K137V, K155E, F158N, P160E, K193R, K194D, N197S, W203Y, D228S, K229Q,R231A, G232P, E240T, E262L, N287F, D290Q, K302L, R303E, E307R, W331L,E345P, K348E, M349T, N352R, D377S, K382L, K384S, E389D, K392L, K393Y,K396M, R397L, S403L, N407Q 314 pA15957 6 S4M, K5L, D6Q, R9I, K10N, K11I,E29R, F30A, K32Q, K34Y, G35N, D36L, E59S, K62R, N63D, K67E, K70D, E71R,K72T, E76R, D77N, K78R, S96A, N101E, K104R, D105Q, K116T, Y135L, K137V,I148V, E151R, P160E, S190E, S191T, K193R, K194A, N197S, W203Y, G216R,K221Q, E262L, D290E, K302L, R303E, E307R, D313P, K318D, W331L, E345P,K348E, M349T, N352R, E380R, K382L, K384S, E389D, K392L, K393A, K396I,R397L, E400R, S403F, N407H 315 pA15958 6 S4P, K5A, D6H, K10E, K11H,S21E, E33N, K34M, D36E, M56W, E59S, K62R, N63D, M66Y, K67E, K70D, E71R,E76R, K78R, K131E, V132T, E136D, K155Q, Y156M, S190E, S191R, K193R,K194E, N197R, Y219G, K238S, E262Q, N287R, K302I, R303E, E307R, K321R,D322T, R325G, L328Y, W331L, E343N, E345P, K348R, M349E, N352R, G361P,K384R, N385P, S403A, N407H, L408D 316 pA15959 6 L3T, S4D, K5H, D6A,K10E, K11I, S21E, E25R, E29R, K32I, D36T, N63R, K67E, K70D, K72E, E75R,E76R, K78L, S96E, N101Q, K104R, D105E, K116T, K131E, P134D, Y135D,K137R, E140R, I148V, E151R, T152Y, A153R, R154Q, K155Q, N157R, F158V,S187R, S190D, S191A, K193R, K194R, N197S, W203F, Y219G, K221Q, D228S,K229R, R231E, G232P, E235R, K238S, E262R, S299A, K318D, E343N, E345P,K348R, M349E, N352R, K353R, I364P, K365R, F366Y, D373E, F376E, E380R,K382L, K384R, E389D, K392L, K393A, K396M, R397L, E400R, L408D 317pA15960 6 S4P, K5A, D6H, K10R, K11R, S21E, E33N, K34M, N63R, K67E, K70D,E71R, E75R, E76R, K78L, S96E, K104R, D105E, K116D, K131E, V132T, P134A,Y135L, E136P, K137R, E147R, I148E, K155Q, Y156M, Q159R, V179I, S190E,S191Y, K193R, K194R, Y219G, D228S, K229Q, G232P, N287R, K302I, R303E,E307R, K321S, D322T, S323G, R325D, L328Y, W331L, E343N, E345P, K348R,M349E, N352R, K353R, I364L, K365S, F366L, F376E, E380R, K382L, N407H,L408D 318 pA15961 6 S4M, K5Q, D6A, R9I, K10A, K11I, S21E, E25R, E29R,F30A, K32Q, E33N, K34Y, G35N, D36T, E59S, K62R, N63D, M66Y, K67R, K70D,K72E, E76R, D77N, K78R, E94L, S96E, T98Q, N101Q, K102Y, K104R, D105E,K116T, P130S, K131E, V132T, E136P, K137R, I148V, E151R, K155E, N157D,F158N, S190E, S191Y, K193R, K194R, N197E, P200M, G216R, D220Q, K221T,D228S, K229Q, R231E, G232P, E235R, K238S, R239S, E240T, E262L, N287R,D290Q, E297L, K302L, R303E, E307R, K318N, K321R, K324E, R325D, E327R,L328Y, W331L, E343N, E345P, K348E, M349Q, N352R, K353E, E356R, N357H,I364P, K365R, D377R, E380N, K382E, K384R, E389D, K392E, K393A, K396Q,R397E, S403F, N4071 319 pA15962 6 K5H, K10E, K11H, S21E, E29R, K32I,E33N, K34M, G35H, Y37P, M56W, E59S, K62R, N63D, M66Y, K67E, K70D, E71R,K72T, E76R, D77N, K78L, S96E, N101Q, K104R, D105E, K116D, K131E, V132T,P134A, Y135L, E136P, K137R, E147R, I148E, T152A, Y156M, Q159R, V179I,S190E, S191Y, K193R, K194R, G216R, K221Q, D228S, K229Q, G232P, E262Q,K318D, K321R, D322T, K324E, R325G, E327R, L328Y, E343N, E345P, K348R,M349E, N352R, D373R, K382L, K384R, K392L, K393A, K396M, R397E, E400R,S403A, N407H, L408D 320 pA15963 6 S4P, K5A, D6H, K10E, K11H, S21E, K32I,K34M, G35H, Y37P, N63R, K67E, K70D, E71R, K72L, E76R, D77N, K78L, E94L,T98Q, N101Q, K102Y, K104R, D105E, K116T, Y135L, E136P, E147R, I148E,K155Q, Y156M, V179I, S190D, S191E, K193R, K194R, K229Q, G232P, E262Q,K318D, K321S, D322T, R325D, L328Y, E343N, E345P, K348R, M349E, N352R,G361P, D363P, I364L, K365S, D373L, K382L, K384E, N385P, D386H, R388E,E389R, K392R, K393A, K396M, R397E, E400R, N407H, L408D 374 pA16624 6C17T 375 pA16625 6 S18N 376 pA16626 6 S18A 377 pA16631 6 Q46W 378pA16632 6 Q46R 379 pA16633 6 D84P 380 pA16634 6 D84A 381 pA16635 6 L89G382 pA16637 6 H45Q 383 pA16638 6 H45A 384 pA16639 6 H45K 385 pA16640 6Q46A 386 pA16643 6 D84G 387 pA16645 6 L89S 388 pA16647 6 H45F 389pA16649 6 Q46S 390 pA16651 6 Q46T 391 pA16653 6 Q92A 392 pA16654 6 Q92M393 pA16655 6 Q92P 394 pA16656 6 Q92D 395 pA16657 6 S123A 396 pA16659 6Q92G 397 pA16660 6 H119N 398 pA16663 6 H119G 399 pA16664 6 H119P 400pA16665 6 C122G 401 pA16668 6 C122E 402 pA16669 6 C122K 403 pA16670 6H119S 404 pA16671 6 S123M 405 pA16672 6 S123R 406 pA16673 6 S123T 407pA16674 6 M124R 408 pA16675 6 M124K 409 pA16678 6 T166M 410 pA16680 6T1661 411 pA16682 6 T166L 412 pA16684 6 D167A 413 pA16685 6 D167V 414pA16686 6 D167T 415 pA16687 6 D167N 416 pA16688 6 V168M 417 pA16689 6V168R 418 pA16691 6 I170K 419 pA16693 6 A171P 420 pA16694 6 I170T 421pA16695 6 I170M 422 pA16705 6 E175K 423 pA16706 6 E176Y 424 pA16707 6E175V 425 pA16708 6 E176F 426 pA16710 6 G173S 427 pA16712 6 E175R 428pA16719 6 G178P 429 pA16720 6 V210P 430 pA16722 6 V210T 431 pA16726 6M212K 432 pA16729 6 M212L 433 pA16730 6 M212R 434 pA16733 6 T249S 435pA16736 6 R339G 436 pA16737 6 R339A 437 pA16738 6 R339T 438 pA16740 6R339E 439 pA16741 6 Y340P 440 pA16742 6 Y340R 441 pA16743 6 Y340H 442pA16744 6 Y53V 443 pA16745 6 Y53S 444 pA16746 6 Y53A

In some embodiments, the polypeptide comprises the amino acid sequenceset forth in SEQ ID NO: 1 is derived from Caldithrix abyssi. In someembodiments, the polypeptide may have a molecular weight of 50.0 kDa to52.0 kDa. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 2 is derived from Anaerolineathermophila. In some embodiments, the polypeptide may have a molecularweight of 46.3 kDa to 48.3 kDa. In some embodiments, the polypeptidecomprises the amino acid sequence set forth in SEQ ID NO: 3 is derivedfrom Thermoanaerobacterium thermosaccharolyticum. In some embodiments,the polypeptide may have a molecular weight of 48.4 kDa to 50.4 kDa. Insome embodiments, the polypeptide comprises the amino acid sequence setforth in SEQ ID NO: 4 is derived from Thermoanaerobacterthermohydrosulfuricus. In some embodiments, the polypeptide may have amolecular weight of 50.0 kDa to 52.0 kDa. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5is derived from Caldicellulosiruptor kronotskyensis. In someembodiments, the polypeptide may have a molecular weight of 48.3 kDa to50.3 kDa. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 6 is derived from Dictyoglomusturgidum. In some embodiments, the polypeptide may have a molecularweight of 46.9 kDa to 48.9 kDa. In some embodiments, the polypeptidecomprises the amino acid sequence set forth in SEQ ID NO: 7 is derivedfrom Caldilinea aerophila. In some embodiments, the polypeptide may havea molecular weight of 44.7 kDa to 46.7 kDa. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO: 8is derived from Rhodothermus marinus. In some embodiments, thepolypeptide may have a molecular weight of 45.9 kDa to 47.9 kDa. In someembodiments, the polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 9 is derived from Methanohalobium evestigatum. In someembodiments, the polypeptide may have a molecular weight of 44.7 kDa to46.7 kDa. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 10 is derived from Thermoanaerobacterthermohydrosulfuricus. In some embodiments, the polypeptide may have amolecular weight of 37.6 kDa to 39.6 kDa. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO: 11is derived from Clostridium cavendishii. In some embodiments, thepolypeptide may have a molecular weight of 48.7 kDa to 50.7 kDa. In someembodiments, the polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 12 is derived from Kosmotoga olearia. In some embodiments,the polypeptide may have a molecular weight of 48.3 kDa to 50.3 kDa. Insome embodiments, the polypeptide comprises the amino acid sequence setforth in SEQ ID NO: 13 is derived from Butyricicoccus pullicaecorum. Insome embodiments, the polypeptide may have a molecular weight of 48.0kDa to 50.0 kDa. In some embodiments, the polypeptide comprises theamino acid sequence set forth in SEQ ID NO: 14 is derived fromClostridium thermobutyricum. In some embodiments, the polypeptide mayhave a molecular weight of 50.6 kDa to 52.6 kDa. In some embodiments,the polypeptide comprises the amino acid sequence set forth in SEQ IDNO: 15 is derived from Litorilinea aerophila. In some embodiments, thepolypeptide may have a molecular weight of 46.5 kDa to 48.5 kDa. In someembodiments, the polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 16 is derived from Enterobacter mori. In some embodiments,the polypeptide may have a molecular weight of 47.5 kDa to 49.5 kDa. Insome embodiments, the polypeptide comprises the amino acid sequence setforth in SEQ ID NO: 17 is derived from Caldisericum exile. In someembodiments, the polypeptide may have a molecular weight of 47.1 kDa to49.1 kDa. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 18 is derived from Dictyoglomusthermophilum. In some embodiments, the polypeptide may have a molecularweight of 46.9 kDa to 48.9 kDa. In some embodiments, the polypeptidecomprises the amino acid sequence set forth in SEQ ID NO: 19 is derivedfrom Rhodothermus marinus. In some embodiments, the polypeptide may havea molecular weight of 45.9 kDa to 47.9 kDa. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO: 20is derived from Rhodothermus profundi. In some embodiments, thepolypeptide may have a molecular weight of 46.3 kDa to 48.3 kDa. In someembodiments, the polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 21 is derived from Caldibacillus debilis. In someembodiments, the polypeptide may have a molecular weight of 47.5 kDa to49.5 kDa. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 22 is derived from Caloramatorquimbayensis. In some embodiments, the polypeptide may have a molecularweight of 46.8 kDa to 48.8 kDa. In some embodiments, the polypeptidecomprises the amino acid sequence set forth in SEQ ID NO: 23 is derivedfrom Methanosalsum zhilinae. In some embodiments, the polypeptide mayhave a molecular weight of 41.6 kDa to 43.6 kDa. In some embodiments,the polypeptide comprises the amino acid sequence set forth in SEQ IDNO: 321 is derived from Pseudothermotoga thermarum. In some embodiments,the polypeptide may have a molecular weight of 56.2 kDa to 58.2 kDa. Insome embodiments, the polypeptide comprises the amino acid sequence setforth in SEQ ID NO: 322 is derived from Pseudothermotoga hypogea. Insome embodiments, the polypeptide may have a molecular weight of 54.8kDa to 56.8 kDa. In some embodiments, the polypeptide comprises theamino acid sequence set forth in SEQ ID NO: 323 is derived fromPseudothermotoga lettingae. In some embodiments, the polypeptide mayhave a molecular weight of 55.9 kDa to 57.9 kDa. In some embodiments,the polypeptide comprises the amino acid sequence set forth in SEQ IDNO: 324 is derived from Rhodothermus marinus. In some embodiments, thepolypeptide may have a molecular weight of 57.5 kDa to 59.5 kDa. In someembodiments, the polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 325 is derived from Geosporobacter subterraneus. In someembodiments, the polypeptide may have a molecular weight of 56.2 kDa to58.2 kDa. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 326 is derived from Melioribacterroseus. In some embodiments, the polypeptide may have a molecular weightof 58.0 kDa to 60.0 kDa. In some embodiments, the polypeptide comprisesthe amino acid sequence set forth in SEQ ID NO: 327 is derived fromLysinibacillus sphaericus. In some embodiments, the polypeptide may havea molecular weight of 55.2 kDa to 57.2 kDa. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:328 is derived from Clostridium stercorarium. In some embodiments, thepolypeptide may have a molecular weight of 56.9 kDa to 58.9 kDa. In someembodiments, the polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 329 is derived from Truepera radiovictrix. In someembodiments, the polypeptide may have a molecular weight of 51.8 kDa to53.8 kDa. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 330 is derived from Dictyoglomusturgidum. In some embodiments, the polypeptide may have a molecularweight of 55.7 kDa to 57.7 kDa. In some embodiments, the polypeptidecomprises the amino acid sequence set forth in SEQ ID NO: 331 is derivedfrom Caldilinea aerophila. In some embodiments, the polypeptide may havea molecular weight of 51.0 kDa to 53.0 kDa. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:332 is derived from Thermoflexus hugenholtzii. In some embodiments, thepolypeptide may have a molecular weight of 51.5 kDa to 53.5 kDa. In someembodiments, the polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 333 is derived from Thermoanaerobacteriumthermosaccharolyticum. In some embodiments, the polypeptide may have amolecular weight of 54.7 kDa to 56.7 kDa. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:334 is derived from Petrotoga mobilis. In some embodiments, thepolypeptide may have a molecular weight of 43.8 kDa to 45.8 kDa. In someembodiments, the polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 335 is derived from Spirochaeta thermophila. In someembodiments, the polypeptide may have a molecular weight of 46.2 kDa to48.2 kDa. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 336 is derived from Thermofilumpendens. In some embodiments, the polypeptide may have a molecularweight of 53.4 kDa to 55.4 kDa. In some embodiments, the polypeptidecomprises the amino acid sequence set forth in SEQ ID NO: 337 is derivedfrom Rhodothermus marinus. In some embodiments, the polypeptide may havea molecular weight of 57.3 kDa to 59.3 kDa. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:338 is derived from Dictyoglomus thermophilum. In some embodiments, thepolypeptide may have a molecular weight of 55.9 kDa to 57.9 kDa. In someembodiments, the polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 339 is derived from Thermoanaerobacter siderophilus. Insome embodiments, the polypeptide may have a molecular weight of 55.0kDa to 57.0 kDa. In some embodiments, the polypeptide comprises theamino acid sequence set forth in SEQ ID NO: 340 is derived fromThermoanaerobacter mathranii. In some embodiments, the polypeptide mayhave a molecular weight of 54.8 kDa to 56.8 kDa. In some embodiments,the polypeptide comprises the amino acid sequence set forth in SEQ IDNO: 341 is derived from Thermoanaerobacter italicus. In someembodiments, the polypeptide may have a molecular weight of 54.9 kDa to56.9 kDa. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 342 is derived fromThermoanaerobacterium thermosaccharolyticum. In some embodiments, thepolypeptide may have a molecular weight of 54.7 kDa to 56.7 kDa. In someembodiments, the polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 343 is derived from Thermoanaerobacteriumthermosaccharolyticum. In some embodiments, the polypeptide may have amolecular weight of 54.7 kDa to 56.7 kDa. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:344 is derived from Thermoanaerobacterium thermosaccharolyticum. In someembodiments, the polypeptide may have a molecular weight of 54.6 kDa to56.6 kDa. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 345 is derived fromThermoanaerobacterium thermosaccharolyticum. In some embodiments, thepolypeptide may have a molecular weight of 54.5 kDa to 56.5 kDa. In someembodiments, the polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 346 is derived from Thermoanaerobacterium xylanolyticum.In some embodiments, the polypeptide may have a molecular weight of 54.2kDa to 56.2 kDa. In some embodiments, the polypeptide comprises theamino acid sequence set forth in SEQ ID NO: 347 is derived fromPetrotoga mobilis. In some embodiments, the polypeptide may have amolecular weight of 43.9 kDa to 45.9 kDa. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:348 is derived from Thermoanaerobacterium saccharolyticum. In someembodiments, the polypeptide may have a molecular weight of 54.0 kDa to56.0 kDa. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 349 is derived from Petrotoga mobilis.In some embodiments, the polypeptide may have a molecular weight of 44.4kDa to 46.4 kDa. In some embodiments, the polypeptide comprises theamino acid sequence set forth in SEQ ID NO: 350 is derived fromSpirochaeta thermophila. In some embodiments, the polypeptide may have amolecular weight of 46.5 kDa to 48.5 kDa. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:351 is derived from Ignisphaera aggregans. In some embodiments, thepolypeptide may have a molecular weight of 29.1 kDa to 31.1 kDa. In someembodiments, the polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 352 is derived from Thermotoga maritima. In someembodiments, the polypeptide may have a molecular weight of 54.6 kDa to56.6 kDa. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 353 is derived from Caldanaerobactersubterraneus. In some embodiments, the polypeptide may have a molecularweight of 55.2 kDa to 57.2 kDa. In some embodiments, the polypeptidecomprises the amino acid sequence set forth in SEQ ID NO: 354 is derivedfrom Mesotoga infera. In some embodiments, the polypeptide may have amolecular weight of 57.1 kDa to 59.1 kDa. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:355 is derived from Thermosinus carboxydivorans. In some embodiments,the polypeptide may have a molecular weight of 54.5 kDa to 56.5 kDa. Insome embodiments, the polypeptide comprises the amino acid sequence setforth in SEQ ID NO: 356 is derived from Halanaerobium congolense. Insome embodiments, the polypeptide may have a molecular weight of 57.5kDa to 59.5 kDa. In some embodiments, the polypeptide comprises theamino acid sequence set forth in SEQ ID NO: 357 is derived fromHalanaerobium congolense. In some embodiments, the polypeptide may havea molecular weight of 57.5 kDa to 59.5 kDa. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:358 is derived from Halanaerobium saccharolyticum. In some embodiments,the polypeptide may have a molecular weight of 57.2 kDa to 59.2 kDa. Insome embodiments, the polypeptide comprises the amino acid sequence setforth in SEQ ID NO: 359 is derived from Gracilibacillus halophilus. Insome embodiments, the polypeptide may have a molecular weight of 56.1kDa to 58.1 kDa. In some embodiments, the polypeptide comprises theamino acid sequence set forth in SEQ ID NO: 360 is derived fromCaldanaerobacter subterraneus. In some embodiments, the polypeptide mayhave a molecular weight of 55.6 kDa to 57.6 kDa. In some embodiments,the polypeptide comprises the amino acid sequence set forth in SEQ IDNO: 361 is derived from Litorilinea aerophila. In some embodiments, thepolypeptide may have a molecular weight of 45.2 kDa to 47.2 kDa. In someembodiments, the polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 362 is derived from Caldanaerobacter subterraneus. In someembodiments, the polypeptide may have a molecular weight of 55.4 kDa to57.4 kDa. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 363 is derived from Caldanaerobactersubterraneus. In some embodiments, the polypeptide may have a molecularweight of 56.3 kDa to 58.3 kDa. In some embodiments, the polypeptidecomprises the amino acid sequence set forth in SEQ ID NO: 364 is derivedfrom Caldicoprobacter faecalis. In some embodiments, the polypeptide mayhave a molecular weight of 54.4 kDa to 56.4 kDa. In some embodiments,the polypeptide comprises the amino acid sequence set forth in SEQ IDNO: 365 is derived from Thermoanaerobacter uzonensis. In someembodiments, the polypeptide may have a molecular weight of 55.2 kDa to57.2 kDa. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 366 is derived from Lactobacillusingluviei. In some embodiments, the polypeptide may have a molecularweight of 60.1 kDa to 62.1 kDa. In some embodiments, the polypeptidecomprises the amino acid sequence set forth in SEQ ID NO: 367 is derivedfrom Petrotoga mexicana. In some embodiments, the polypeptide may have amolecular weight of 44.4 kDa to 46.4 kDa. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:368 is derived from Defluviitoga tunisiensis. In some embodiments, thepolypeptide may have a molecular weight of 44.0 kDa to 46.0 kDa. In someembodiments, the polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 369 is derived from Petrotoga miotherma. In someembodiments, the polypeptide may have a molecular weight of 43.8 kDa to45.8 kDa. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 370 is derived from Petrotoga olearia.In some embodiments, the polypeptide may have a molecular weight of 43.7kDa to 45.7 kDa. In some embodiments, the polypeptide comprises theamino acid sequence set forth in SEQ ID NO: 371 is derived fromThermophagus xiamenensis. In some embodiments, the polypeptide may havea molecular weight of 46.4 kDa to 48.4 kDa. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:372 is derived from Treponema caldarium. In some embodiments, thepolypeptide may have a molecular weight of 45.9 kDa to 47.9 kDa. In someembodiments, the polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 373 is derived from Thermofilum uzonense. In someembodiments, the polypeptide may have a molecular weight of 52.9 kDa to54.9 kDa.

Polypeptides having D-fructose C4-epimerase activity according to thedisclosure can be provided by means of recombinant DNA technologymethods allowing it to be produced by cultivating in a culturing mediuman appropriate host organism cell comprising a gene coding for theD-fructose C4-epimerase, and recovering the enzyme from the cells and/orthe culturing medium. Examples of host cells for transformation includeEscherichia. coli, (hereinafter referred to as E. coli), Corynebacterumglutamicum, Aspergillus oryzae, Pichia pastoris, or Bacillus subtilis,and the like. Examples of transformed E. coli microorganisms may includeE. coli NEBT7EL-pA06233, NEBT7EL-pA06234, NEBT7EL-pA06235,NEBT7EL-pA06236, NEBT7EL-pA06237, NEBT7EL-pA06238, NEBT7EL-pA06239,NEBT7EL-pA06240, NEBT7EL-pA06241, NEBT7EL-pA07068, NEBT7EL-pA07069,NEBT7EL-pA07070, NEBT7EL-pA07071, NEBT7EL-pA07072, NEBT7EL-pA07073,NEBT7EL-pA07074, NEBT7EL-pA07075, NEBT7EL-pA07076, NEBT7EL-pA07077,NEBT7EL-pA07078, NEBT7EL-pA07079, NEBT7EL-pA07080, NEBT7EL-pA07081,NEBT7EL-pA06242, NEBT7EL-pA06243, NEBT7EL-pA06246, NEBT7EL-pA06247,NEBT7EL-pA06248, NEBT7EL-pA06249, NEBT7EL-pA06250, NEBT7EL-pA06252,NEBT7EL-pA06253, NEBT7EL-pA06254, NEBT7EL-pA06255, NEBT7EL-pA06256,NEBT7EL-pA06257, NEBT7EL-pA06261, NEBT7EL-pA06265, NEBT7EL-pA06266,NEBT7EL-pA06267, NEBT7EL-pA06268, NEBT7EL-pA06270, NEBT7EL-pA06271,NEBT7EL-pA06272, NEBT7EL-pA06273, NEBT7EL-pA06274, NEBT7EL-pA07082,NEBT7EL-pA07083, NEBT7EL-pA07084, NEBT7EL-pA07085, NEBT7EL-pA07086,NEBT7EL-pA07087, NEBT7EL-pA07088, NEBT7EL-pA07089, NEBT7EL-pA07090,NEBT7EL-pA07091, NEBT7EL-pA07092, NEBT7EL-pA07094, NEBT7EL-pA07095,NEBT7EL-pA07096, NEBT7EL-pA07097, NEBT7EL-pA07098, NEBT7EL-pA07099,NEBT7EL-pA07100, and NEB T7EL-pA07101.

In some embodiments, the polypeptide comprises the amino acid sequenceset forth in SEQ ID NO: 1. In some embodiments, the polypeptidecomprises the amino acid sequence set forth in SEQ ID NO: 2. In someembodiments, the polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 3. In some embodiments, the polypeptide comprises theamino acid sequence set forth in SEQ ID NO: 4. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5.In some embodiments, the polypeptide comprises the amino acid sequenceset forth in SEQ ID NO: 6. In some embodiments, the polypeptidecomprises the amino acid sequence set forth in SEQ ID NO: 7. In someembodiments, the polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 8. In some embodiments, the polypeptide comprises theamino acid sequence set forth in SEQ ID NO: 9. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 11. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:12. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 13. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:14. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 15. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:16. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 17. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:18. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 19. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:20. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 21. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:22. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 23. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:321. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 322. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:323. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 324. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:325. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 326. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:327. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 328. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:329. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 330. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:331. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 332. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:333. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 334. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:335. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 336. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:337. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 338. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:339. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 340. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:341. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 342. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:343. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 344. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:345. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 346. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:347. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 348. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:349. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 350. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:351. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 352. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:353. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 354. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:355. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 356. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:357. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 358. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:359. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 360. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:361. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 362. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:363. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 364. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:365. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 366. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:367. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 368. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:369. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 370. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:371. In some embodiments, the polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 372. In some embodiments, thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO:373.

TABLE 3 Native Sequences SEQ Plasmid NO. ID Sequence Organism 1 pA06233MSLHPLNKLIERHKKGTPVGIYSVCSANPFVL Caldithrix abyssiKAAMLQAQKDQSLLLIEATSNQVDQFGGYTG MRPEDFKTMTLELAAENNYDPQGLILGGDHLGPNRWTKLSASRAMDYAREQIAAYVKAGFSK IHLDATMPLQNDATDSAGRLPVETIAQRTAELCAVAEQTYRQSDQLFPPPVYIVGSDVPIPGGA QEALNQIHITEVKEVQQTIDHVRRAFEKNGLEAAYERVCAVVVQPGVEFADQIVFEYAPDRAA ALKDFIESHSQLVYEAHSTDYQTAPLLRQMVKDHFAILKVGPALTFALREAIFALAFMEKELLPL HRALKPSAILETLDQTMDKNPAYWQKHYGGTKEEVRFAQRFSLSDRIRYYWPFPKVQKALRQL LKNLQQISIPLTLVSQFMPEEYQRIRQGTLTNDPQALILNKIQSVLKQYAEATQIQNSLTFTQNQN SLAMERL 2 pA06234MMFGSPAPLLDMVTAQKQGMARGIPSICSAH AnaerolineaPVVLSAACHLARRSGAPLLIETTCNQVNHQGG thermophilaYSGMTPADFVRFLREILEREGIPPQQVILGGDH LGPYPWRKEPAETAIAQALEMVRAYVQAGYTKIHLDASMPCADDDPERPLPLERIARRAAQLC AAAEAAAGAVQPVYVIGSEVPPPGGAQGQEARLHVTTPQEAQAALDAFREAFLQAGLTPVWE RVIALVVQPGVEFGVDSIHAYQREAARPLKTFIEGVPGMVYEAHSTDYQTRASLRALVEDHFSIL KVGPALTFAYREAVFALEHIEREILGRQDMPLSRLSEVLDEVMLNDPRHWQGYFAGAPAEQALA RRYSFSDRIRYYWHHPAAQEAVRRLLANLIETPPPLSLLSQYLPREYEMVRAGEISSHPQDLIRA HIQHTLEDYAAACG 3 pA06235MAKEHPLKELVNKQKSGISEGIVSICSSNEFVIE ThermoanaerobacteriumASMERALTNGDYVLIESTANQVNQYGGYIGM thermosaccharolyticumTPIEFKKFVFSIAKKVDFPLDKLILGGDHLGPLI WKNESSNLALAKASELIKEYVLAGYTKIHIDTSMRLKDDTDFNTEIIAQRSAVLLKAAENAYMEL NKNNKNVLHPVYVIGSEVPIPGGSQGSDESLQITDAKDFENTVEIFKDVFSKYGLINEWENIVAF VVQPGVEFGNDFVHEYKRDEAKELTDALKNYKTFVFEGHSTDYQTRESLKQMVEDGIAILKVG PALTFALREALIALNNIENELLNNVDSIKLSNFTNVLVSEMINNPEHWKNHYFGDDARKKFLCKY SYSDRCRYYLPTRNVKNSLNLLIRNLENVKIPMTLISQFMPLQYDNIRRGLIKNEPISLIKNAIMN RLNDYYYAIKP 4 pA06236MNTEHPLKNVVKLQKKGIPIGIYSVCSANEIVI ThermoanaerobacterQVAMEKALSMDSYVLIEATANQVNQYGGYT thermohydrosulfuricusNMKPIDFRDFVYSIAKRINFPENRIILGGDHLGP LPWKNQQAKKAMEEAKELVKQFVMAGFTKIHVDTSMLLGDDNINIKLDTETIAERGAILVSVA ERAFEELKKFNPYALHPVYVIGSEVPVPGGSQKENNNEIQVTKPTDFEETVEVYKSTFYKYGLG NAWEDVVAVVVQAGVEFGVEDIHEYDHQQAENLVSALKKYPNLVFEAHSTDYQPAKLLKEM VRDGFAILKVGPELTFALREGLFALNIIEKELFKDNHDIEMSNFIDILDTAMLNNPKYWEQYYYG DDNKIRIARKYSYSDRCRYYLIENEVRASMSRLFKNLTNVEIPLTLISQYMPIQYEKIRMGLLKN DPENLVKDKIGNCIDKYLYATNPTSGEFKLI 5pA06237 MSPQNPLIGLFKNREKEFKGIISVCSSNEIVLEA CaldicellulosiruptorVLKRMKDTNLPIIIEATANQVNQFGGYSGLTPS kronotskyensisQFKERVIKIAQKVDFPLERIILGGDHLGPFVWR DQEPEIAMEYAKQMIKEYIKAGFTKIHIDTSMPLKGENSIDDEIIAKRTAVLCRIAEECFEKISINNP YITRPVYVIGADVPPPGGESSICQTITTKDELERSLEYFKEAFKKEGIEHVFDYVVAVVANFGVEF GSDEIVDFDMEKVKPLKELLAKYNIVFEGHSTDYQTKENLKRMVECGIAILKVGPALTFTLREA LVALSHIEEEIYSNEKEKLSRFREVLLNTMLTCKDHWSKYFDENDKLIKSKLLYSYLDRWRYYF ENESVKSAVYSLIGNLENVKIPPWLVSQYFPSQYQKMRKKDLKNGAADLILDKIGEVIDHYVYA VKE 6 pA06238MWLSKDYLRKKGVYSICSSNSYVIEASIEFAKE DictyoglomusKGDYILIEATPHQVNQFGGYSGMTPEDFKNFV turgidumMKIAKEKGLEEDKIILGGDHLGPLPWQDEPSPT AMNKAKDLIRAFVESGYKKIHIDCSMPLSDDPKVLPYEKIAERTRELFEIAEETARKYNFQPVYV VGTDVPIAGGGEEEGVTSVEDFRSAISSLKKYFNDVPNIWDRVVGFVIMLGIGFSYDKVFEYDRD KVRGILEEVKREDLFVEGHSTDYQARYALRNMVEDGVRILKVGPALTAAFRRGVFLLSNIEDEI IPERERSNIKRVILETMLRDDRYWRKYYKDSKRLELDIWYNLLDRIRYYWEYEDVKMVLNKLF ENFSEGVDIKFIYQYFYDSYFDVREGKMKNDPRELIKKEIKRVLEDYSYAINL 7 pA06239 MSTLRHIILRLIELREREQIHLTLLAVCPNSAAVCaldilinea LEAAVKVAARCHTPMLFAATLNQVDRDGGY aerophilaTGWTPAQFVAEMRRYAVRYGCTTPLYPCLDH GGPWLKDRHAQEKLPLDQAMHEVKLSLTACLEAGYALLHIDPTVDRTLPPGEAPLVPIVVERTV ELIEHAEQERQRLNLPAVAYEVGTEEVHGGLVNFDNFVAFLDLLKARLEQRALMHAWPAFVVA QVGTDLHTTYFDPSAAQRLTEIVRPTGALLKGHYTDWVENPADYPRVGMGGANVGPEFTAAE FEALEALERREQRLCANRKLQPACFLAALEEAVVASDRWRKWLQPDEIGKPFAELTPARRRWL VQTGARYVWTAPKVIAAREQLYAHLSLVQADPHAYVVESVARSIERYIDAFNLYDAATLLG 8 pA06240 MQAQALLTVPFDRVATHARGFVGWVAELLQRhodothermus GPLAYQHTLLAVCPNSEAVTRAALEAAAEAN marinusAPLLFAATLNQVDLDGGYTGWTPATLARFVA DELARLDLHIPVVLGLDHGGPWKKDLHARNRLSFEETFQAVLRAIEACLDAGYGLLHLDPTVD LELSPGTPVPIPRIVERSVALLRHAETYRLRRNLPPVAYEVGTEEVGGGLQAEARMAEFLDRLWT ALDREGLPHPVFVVGDIGTRLDTRTFDFERARRLDALVRRYGALIKGHYTDDVDRLDLYPKAG IGGANVGPGLAAIEFEALEALVEEARRRGLSVTFDQAIRRAVVESGRWTKWLQPEEKGQPFDA LDPERQRWLVATGSRYVWTHPAVLQARRELYEALAPWLDADAFVRTRIKARLMDYFRAFNLIH FNERLQAFLPE 9 pA06241MTDEDFEPICEISEQFRNYCNQMLESEYDPKPS MethanohalobiumKYIFNILQNQKTIVMAANPRIGLVTRGILRAAK evestigatumDADAPIILELARSECNLENGYTGLYPSDFSEQC YQAAKDVGYDIWALHADHIGIKKGDREDIEKTKELVKAQIDAGYTSFAIDASHLFNFQGGDLR EELKDNIDATTEIAKFIEEQMDDREYGLEVEVGEIGREDEHGRVLTNPEEAVTFIKALNENGVY PQVLAIANGSAHGNTYDSQGRLIEQVSIDIPQTIKVAQALKENNLKVRIAQHGITGTPRELIHDHF PHGEIIKGNVGTFYMNLVWDAFKLFEPELYNDIWNWTVENYKQKSPDKTDSEIFGKYSKFAIKQ FFDRIYSVNEDTKRAIDAMAYAETLYFLKSFNAERTASIVRDGIK 10 pA07068 MNTEHPLKNVVKLQKKGIPIGIYSVCSANEIVIThermoanaerobacter QVAMEKALSMDSYVLIEATANQVNQYGGYT thermohydrosulfuricusNMKPIDFRDFVYSIAKRINFPENRIILGGDHLGP LPWKNQQAKKAMEEAKELVKQFVMAGFTKIHVDTSMFLGDDNINIKLDTETIAERGAILVSVA ERAFEELKKSNPYALHPVYVIGSEVPVPGGSQKENNNEIQVTKPADFEETVEVYKSTFYKYGLG NAWEDVVAVVVQPGVEFGVENIHEYDHQQAENLVSALKKYPNLVFEAHSTDYQPAKLLKEM VRDGFAILKVGPELTFALREGLFALNIIEKELFKDNHDIEMSNFIDILDTAMLNNPKY 11 pA07069 MQRNYLLDIVEAQNNGIHKGIYSACSANEYVIClostridium EAAMERAKNTNEYVLIEATANQVNQYGGYTG cavendishiiMKPIDFKNFVYDIADKINFDKDKIILGGDHLGP LTWSKETEKEAMAKSHELVKEYVMAGFTKIHLDTSMYLADDDRSKKLATEVIARRGAELCKT AEESFKALKERNSMAVAPVYIVGSEVPIPGGIQDEEEGIQVTKPEDFLETVKVYKAEFKDKGIDE VWNRVIGVVVQPGVEFGDESVHEYNREKAEKLVNSLRGVKGIVFEGHSTDYQTKTKLKEMVE DGIAILKVGPALTYGLREALFALNHIENEIFKYRADIKLSNFINVLETSMVEEPTHWKQHYHGDA EDIKYAMRYSYSDRCRYYMPTEAVNKAMNILIENLESVEIPLTIIDQYMPMQYKKIREGLIQNKP KELIKDRIGDYIDDYLYALR 12 pA07070MKKHPLQDIVSLQKQGIPKGVFSVCSANRFVIE Kosmotoga oleariaTTLEYAKMKGTTVLIEATCNQVNQFGGYTGM TPADFREMVFSIAEDIGLPKNKIILGGDHLGPNPWKGQPSDQAMRNAIEMIREYAKAGFTKLHL DASMRLADDPGNENEPLNPEVIAERTALLCLEAERAFKESAGSLRPVYVIGTDVPPPGGAQNEG KSIHVTSVQDFERTVELTKKAFFDHGLYEAWGRVIAVVVQPGVEFGNEHIFEYDRNRARELTEAI KKHPNIVFEGHSTDYQTAKALKEMVEDGVAILKVGPALTFALREAFFALSSIEKELFYDTPGLCS NFVEVVERAMLDNPKHWEKYYQGEERENRLARKYSFLDRLRYYWNLPEVRTAVNKLITNLET KEIPLTLISQFMPMQYQKIRNGLLRKDPISLIKDRITLVLDDYYFATHPEC 13 pA07071 MNHNPLKKIVELQKQGKNVGIYSVCSANGYVIButyricicoccus EAALKRGKSDGSCVLIESTANQCDQNGGYTG pullicaecorumMTPLDFKNFVLGIADKVGFDPKRLFLGGDHLG PLTFAGMDEAQAMENAEELIRHYVGAGFTKIHIDTSMKVASDDPNTRLSDETIAKRGARLARVA QDTYHKLLESDPDAIAPVYIVGSEVPIPGGAVGAVDQGVQVTKVEDFKNTVATFEKAFREQGLD EAWDNVIGVVVQPGVEEKDSGCTEYDREKAKDLMASIQEFPNLVFEGHSTDYQTKIKLRELVED GVGILKVGPALTFAMREGMFALENIEKELIYGTDITPSGFQDALEAEMLKEGKHWRKHYQGTE LELRMKRKYSFSDRCRYYMPTPAVEAAKERLISNLRTLGIPLNLLSQFMPIQYTKVREGLLVNDP VELVEDRIINTIDEYLYATHQKELL 14 pA07072MLLKVKEHPIRELVNRYKNGENVGIFSVCTSN ClostridiumEYVIEAAMERVIDKDLDLLIESTANQVNQDGG thermobutyricumYTGMQPKDFVNYVYKIADKVNFPKDRIILGGD HLGPLTWTKLVQEEAMEKAKVLIRDYVLAGFTKIHIDTSMPIYDDLEKGVFGDDLIAERAAILC NVAEIAYRELLKTNEDAIHPIYVVGSEVPVPGGVQAEEAEEEIENGIKVTRVEDFKNTVEVFKKK FKEHGVEEAFNYVVGVVVQPGVEFSSDTVWKYEREKAKDLSKALKEYDNLVFEAHSTDYQSP KSLREMVLDGFNILKVGPALTFGFREAAFALNKIEEEMFRFRPDIEESRFIQTLDYNMVKHPENW IKHYSGTSENIRFSRMYSLSDRCRYYMPNEEVEYSFNKMINNLDKEEIPTALISQYMHNQYKKV RDGELKPKGLNLLKDFIGEYVDDYIFAVEDK 15pA07073 MYPVLENILRAQQQGEALGIPSICSAHPFVLEA LitorilineaTFRHALTTGRTVLIESTCNQVNQHGGYTGMTP aerophilaGDFVAYVAALADRLHFPRERILLGGDHLGPNP WRDRPADQALNQARILVQEYVRAGYGKIHLDASMACGGDPADAPLDKAVAAERAAALAEAA EAAFQRMGSGTPPCYVIGTEVPPPGGAQGDDMPLAITAPREVAETIELTQAAFRRRGLEAAWE RVIAVVVQPGVEFGDEQVHPYDRAAAAGLARAIEPYGRLVYEAHSTDYQTRQALRDLVADHF AILKVGPALTFAFREAVFALAAVEEEWLAGQAGVVLSRLREELEAAMIQDPTHWRGYYRGDER HQRLARRYSYSDRARYYWPRPSVQAALERLLHNLEAAPPPLTLLSQYLPVQYWSVREGLLEPT PRSLIVDKIIQVLNDYTWACGG 16 pA07074MERKVKHLTHMVEQHKRGNANGIYAVCSAH Enterobacter mori.PLVLEAAIRYAQSHQTPLLIEATSNQVDQFGG YTGMTPEDFYGFVCCLAESLDFPTSQLILGGDHLGPNRWQNLPAQQAMANADDLIKSYVAAG FKKIHLDCSMSCEDDPVPLTDAIVAERAARLAKIAEATCREQFGVTDLVYVIGTEVPVPGGAHE TLTELEVTTPDAARATLEAHRHALEKEGLNDIWPRIIGLVVQPGVEFDHAHVCDYQPHKAVAL SKMVEAYDTLLFEAHSTDYQTPQALRQLVKDHFAILKVGPALTFALREALFSLAAIEEELLPAK ASSGLRHVLENVMLDRPEYWQSHYHGDGNARRLARGYSYSDRVRYYWPDSQIDDAFERLVR NLADEPIPLPLISQYLPLQYGKVREGALKSTPRELIIDHIQDILQQYHAACEGVTTQNA 17 pA07075 MWLDSNFLKNRGIFSICSSNENVLDASIEFAKECaldisericum exile KEDFLLIEATCHQVNQFGGYTKMTPESFSKKIFKKAEEMNFNPERLLLGGDHLGPEPWKNENAD TAMDKAKQLVIEFVKNGFNKIHLDCSMPLKGDSDFSTTLVADREAELCAVAEETYEKYGGNRP VYVVGTEVPAPGGSTNEVPEVTSIEELDEMIEELQNAFLRLGLKNAWDRVIAIVVRLGIGFGGDS VSEYESEKTKELCTYLSRYYPSLYFEAHSTDYQTAGSLKQMVKDGIRILKVGPALTDAYRRGM FALNFIEKESIDEEKQSRLVENVLKVMDEYPRYWEDYYNSVGKTLRLDQMYSYFDRIRYYWG FEEVEKSKNRLIENLKDMQMNLIRQYLPEQYEKIRENKLNKDPRALINYEIKKVLNDYQKSVILE 18 pA07076MWLSKDYLRKKGVYSICSSNPYVIEASVEFAK DictyoglomusEKNDYILIEATPHQINQFGGYSGMTPEDFKNFV thermophilumMGIIKEKGIEEDRVILGGDHLGPLPWQDEPSSS AMKKAKDLIRAFVESGYKKIHLDCSMSLSDDPVVLSPEKIAERERELLEVAEETARKYNFQPVY VVGTDVPVAGGGEEEGITSVEDFRVAISSLKKYFEDVPRIWDRIIGFVIMLGIGFNYEKVFEYDRI KVRKILEEVKKENLFVEGHSTDYQTKRALRDMVEDGVRILKVGPALTASFRRGVFLLSSIEDEL ISEDKRSNIKKVVLETMLKDDKYWRKYYKDSERLELDIWYNLLDRIRYYWEYKEIKIALNRLFE NFSEGVDIRYIYQYFYDSYFKVREGKIRNDPRELIKNEIKKVLEDYHYAVNL 19 pA07077 MQAQALLTVPFDRVATHARGFVGWVAELLLRhodothermus GPLAHQHTLLAVCPNSEAVTRAALEAAAEVN marinusAPLLFAATLNQVDLDGGYTGWTPATLARFVA DELARLDLHIPVVLGLDHGGPWKKDLHARNRLSFAETVQAVLRAIEACLDAGYGLLHLDPTVD LELPPGTPVPIPRIVERTVALLRHAETYRLRRNLPPVAYEVGTEEVGGGLQAEARMAEFLDRLW TALDREGLPHPIFVVGDIGTRLDTRTFDFERACRLDALVRRYGALIKGHYTDDVDRLDLYPKAG IGGANVGPGLAAIEFEALEVLVDEARRRGLSVTFDQAIRRAVVESGRWTKWLQPEEKGRPFEAL DPERQRWLVATGSRYVWTHPAVLQARRELYEALAPWLDADAFVRERIKARLMDYFRAFNLIHF NERLQAFLPE 20 pA07078MQAHVLLAPSFEQLADHRHGFVGWLVDLLRG RhodothermusPLAYRHTLLAVCPNSEAVTRAALEAAREANAP profundiLFFAATLNQVDLDGGYTGWTPATLARFVADE RIRLGLRAPVVLGLDHGGPWKKDWHVRNRLPYEATLQAVLRAIEACLDAGYGLLHLDPTVDLE LPPGTPVPIPRIVERTVALLQHAETYRQQRRLPPVAYEVGTEEVGGGLQAEARMAEFLDRLWTV LDREGLPRPVFVVGDIGTRLDTHTFDFERARRLDALVRRYGALIKGHYTDGVDRLDLYPQAGI GGANVGPGLAAIEFEALEALVAEAHRRKLPVTFDRTIRQAVIESGRWQKWLRPEEKGRPFEALP PERQRWLVATGSRYVWTHPAVRQARHQLYQVLAPWLDADAFVRARIKARLMDYFRAFNLIGF NERLQAFLPN 21 pA07079MAKIPIQSAVKALLELQDEGKGGTLLGIGPMS CaldibacillusTNVLQASFELARDYDFPLMFIASRNQVDLDEL debilis. GGGYVNGWNQYTFVQAIREMAELTGFDGLYYVCRDHGGPWQRDKERNDHLPVEEAMALGK KSYLADIEAGFDLLMIDPTKDPFEIGKVIPLEKVIERTVELIEFCEKERQARDLPEIGYEVGTEET NGGLTSTETYETFILRLQEELGRRDLPMPTFIVGQTGTLVRKTEQAGRFSFENAADLAKMAKKY GVGLKEHNGDYLDDVILLAHIPSQITATNVAPQYGTEETRALLKLAKLEEKLKEQGLIGQPSKV KDVLLYHSIKSERWRKWMVGSQRELSVEEIVKDEELSTEILDIAGHYTFNIDEVKEEINKLYRN LSKAHIDGQRFVVDHIKRSIRNYVECFNLKGLTSRIKEKLNGSKNA 22 pA07080 MKKISIFEIVKASLNMKGKDKATLLGIGPMSK CaloramatorTLIKASMILAKEKDFPLMFIASRNQVDLKELGG quimbayensisGYVCNWDQKSFASDIKKIAEEVGFNGLYFLCR DHGGPWQRDNERNAHLPENEAMELGKKSYLEDLINGFDLLHIDPTKDPYIVGKTVPMEIVLKR TIELIEYVERERKERNLPPISYEVGTEETNGGLTSEEAYETFIKTLIEELDKRNLPKPSFIVGQTGTL TRLTENVGNFNTKNSKKLADIAKKYSVGLKEHNGDYLDEAILLEHPALGITAMNVAPEFGSVE TQAYLKLIEVENNLYEHGIISKKSNLEKVIKEEAVKSLRWKKWMVGDKVNLSIEEVLSDKDLT DLITEISGHYTFNNERVKCEIQLMFDNLNKAGVDGEKYVINKIKDSIDKYIKYFNLEGFTTKVLS NV 23 pA07081MTDTNYKAKPGSLLFESLMDKETIILAINPRISL MethanosalsumLNKGILKAAKDMDAPIILELAKSECNLEGGYT zhilinaeGFTPSEFSKRAYESAEEIGIDIWSLHADHIGIKK GTDEEIESIKKLVKAQIDAGYTSFAIDASHLFNFRGGNLREELKDNIEATTKVARFIDEQMEDRDY GLEVEVGEIGREDEYGRVLTQPEEAVTFIKALNENGVYPQVLAIANGSAHGNTYDEYGHLIEQ VSIDIPQTMAVARALRDNNLNVRIAQHGITGTPIEMIHNHFPHGDIIKGNVGTFYLNLVWDVLKV FEPQLYGDIWDWTIENFSEKYPDKSENEIFGKYSKYAIKEFFDRIYSVGDDTIRAVESRAYADTLV FLKAFKAAGMAEHVRKNL 321 pA06242MKLLEEFLKAFPGRFKVYGSSLRIITDSYFFLGNDGKEKLLF PseudothermotogaVVGKKGICQLFDGQKIGQIGSNDVLMCKKTHENLLALRKI thermarumINLNPTTINKKASFGFGDRIGLATPAHAKVAKDFEVFPIFAQQSVRELSRTGRTYKDVLDDAVWGVFESGYNFEFGADADHVKEIEDLEKASNEGFTMYTVDPSDHIKDVSKLSQKEFQSLYQDNKIRRELEMRYVGKLYKFKDFEFRMTDEEFAEIFVTYIDAIEHVCKCYDVLKAKGKPFDFEVSIDETAVPTTPLAHIFIVKELRRRGIDFKTLALRFSGEWQKGIDYIGDMEMFRKEIITHSKISKELGGYKLSLHSGSDKFSVYPIFSEATEGEFHVKTAGTNYLEAIRVVAVKDPELYREIHKFALTKFEQDRKSYHVTTDLSKIPDVDKMKNEELVKLLDMPDSRQLIHITYGSVLTAKDENGRWLFKERILKVLQENEDLHYDFVEKHMRKHLS LLGLERRIEK 322 pA06243MFAEFQHLTRGKFVPYATSLRKSTDATFFLVRDELDKYLI PseudothermotogaVIGKKGICELFEGQKIGEIDRQDVVLCAKNDRNCQSLMSL hypogeaFPSLKPQICNAKLSFGFGDRLGVATAAHAQCVQKEKLFPIFAQQSVREISRTERNWLDVLHSAVWGVFESGYDGPFGADADHVKKIEDLESAARAGYTMFTIDPSDHVKDPAKFDKRELVRFYEEHPMRRTLEMKYIGKSFTVLGEKLTFDEENFAEVFVTYIDAIEHVEKCYRALRAVCKTSFDLEVSIDETSVPTTSLAHIFFVQELVRRGVEFRTLALRFPGEWQKGIDYVGDIDLFSENLDKHVAIVKMFTGYRLSLHSGSDKFSVYPILAEKTDRTIHVKTAGTSYLEAIRVVAKFAPDLYRQIHKYALSRFDQDKASYHVTTELSKIPDVDKLEDSELPSLLDQPDSRQLIHITYGSVLTAKKEGRSLFKDRIMRVLFEHEAEHYDFLKKHLGKHIQ LLGV 323 pA06244MAENIVEKFEKLFKGKYKIYYSSIRKLEKSFFFMIRDQKQK PseudothermotogaYLISIAKKRICEKFEGKKIGRINDLDILMCPTNDYNCKVIRTL lettingaeFNINPSVCKKNTSFGFGDRLGLATPAHTTLINKYDVFPVLAQQSVRELSRTHRNFKDVLDSAIWGIFESGYEGEFGADADHVKDINDLMQAAYEGYSMYTVDPSDHVKNIDKINQGELVEFYKSHPLRKEIEMIYSGKVFSFEKSKFTMEDKELFRIFVTYVDAIEHVVKCYEAIKNTKKNFDFEVSIDETSIPTSPLAHIFIVHELRRRGVDFQTLALRFVGQWQKAIDYIGDLSVLESELSMHCEIVKSLSGYRLSLHSGSDKFSVYRIFTHYCDGKLHVKTAGTSYLEAIRTVAEASPSLYRNIHKYALTCFEKDNTSYHVTADINKIPDVDNVEDSKVVNLLDIPEVRQLIHITYGSVLTEKINGKYLFRDEIYRILHENEFLHYKRIRDHLGKHLELLKN 324 pA06245MVTVLQTLLQRPRPLAEIDRTSLARFLTDVIRQQVYPTSLE RhodothermusPTSEGVFFLARDGREKRLGILSEAGLHDFEGVRHQLSLDG marinusRTLIFQSCPLTAANARALRRHIPWTAPRPLGLRASVGCGDRLGLATPGHVRAVRKHKLAPVFAQQSIREMTRTGRTPQQVLDEAMWGVFQEGWRQGYGADADHLKTEEDADRCIEAGFTFFTIDPSAYVDNEVDTADAATLEAKVAALPWEALETTLADLRRAYLGQHFQVGPYELSFEERTLMQALAKYGGAIAHTARMYRHIAGRMGNRPFELEMSVDETEVPTSPAEHFFVARELQRLGVRWISLAPRFVGRLEKGVDYIGDLEEFEAHLKLHVAIARTLGPYKLSLHSGSDKFALYPLFARHAGELFHLKTAGTSYLEALRAVAELDPPLFREILDFARDRYETDRATYHVSALLERVPKASDVPDDALPALLEQFDTRQVLHVTFGSVLTATDADGRPRFRDRLLAVLQENEETYYRLLEAHFDRH LAPFDAE 325 pA06246MDIYEKIAAALKDNRHNIQLDGVKIYPQSYVEVDMVKMI GeosporobacterMVKAAEKKVILAQGSGPLFQELEGEAYDDYKVCNCSHLN subterraneusRVVLNKYLPYTKPSAFGKEIATIGLGDRLGIASPGHIQAVKGREIRPILAQQSIRELNLTNRTYRNVLDAACFAVFQEGYKDGFGADGDHLKTEEDIQSALDLGFTMITLDCSEMIDNTIDKLTDTEVEEKYYQLPQSVRERYETRYLDKCFELRNSKICFSKENLMKNVLIYGAAADFIVAIYEKQIKHRDQKIDFEVSIDETVTPTTPEAHYFVAREIYDRQVDINSMAPRFCGEFQKGIDYIGDIHQFEKEFQVHAEIADHFGYKISIHSGSDKFSVFQTIGRYTEGRFHVKTAGTNWLEAVRVVAEKNPNLYRKMHQYALEHFDEARAYYHVTTDIEGIVPLEKVNDHELSQYMNENNARQLLHITYGILLQAKDASGQYLFREDFFYTLEQHEAE YDEALRKHIGRHLEQLGK 326 pA06247MEMQKLYEEVENKNIVKNDLVDLTIGESLKIKAYPLSVLK MelioribacterKDDAFFFIGKENYDKFLFVISAGKENGLLNEFEGELIDAGK roseusDVTVKKCNLSTKNRKAVQKIFPHTAPIVLGLCNSFGCGDRLGVANAGHIRAIKQSNFRPILAQQSIRELTRTNRTPDDVMDAAVWAVLQEGYKDGFGSDADHLKTFEDIDLMLNAGFTMFTFDPSEHVDNEADNYSEDQLKQKLGEIDWSGLQDTSADAAKRYVDMTFNISERLSLTIQESDFLRAYAKYGNAIAHIKKMYDYLASKADKDTFEIEVSVDETESVTSPFEHFFFANELNRLGVKYVSLAPRFIGDFEKGIDYKGDLNVFKTEYEKHLDITKYFGSYKISLHSGSDKFSAYRVIGSLKGAYTHVKTAGTSYLEALRVVAAKEPALFRDILDFCRDLYETEKRSYHVSADINKVKPANQYSDTELIELFNQNDTRQVLHVTFGKVLTEKDSSGHFLFKDKIMKCLVENEESHYEFLEKHFLKHLECFK 327 pA06248MKQFLPAIELLAKGELPSNSNQIKVYEKSYTVEGNVHLLM LysinibacillusVKNSGEKFILATGEGAIFDELTGTDVDGKGKACPLTYENR sphaericusLVLNKYFDYTVPQAFGTEIATIGLGDRLGLASPGHIETVREKNIKPVLAQQSIRELTLTNRSMNDMLDAAAFAVFQEGYKGGYGADGDHIKEESDIQYALSLGASMITLDCSDHIDNTIEKASPEVLDEKFNALSEVVKQRYMEQYLGKTFEVNGLTLTLDETELKKNVLLYDKAIDYTTHVYNEYISKENRAIDFEISIDETETVTSPISHFFVANELINRGVKVVSLAPRFCGEFQKGIDYIGDVEQFEVELREHALIAEHFGYKLSIHSGSDKFMVFPIIAEYTKGVFHVKTAGTNWLEAIRVIAATNPDLYRRMHVFALENFEEALKYYHVTPDLNSFEKLENVEDAKLPEYMNNDAARQLFHVTYGLLLTAKGENDTFLFRDEFFKTLDKYEEEYRDAL VSHIGKHIELLGL 328 pA06249MGNWKDFVKDFCTKEKNIEVLRAEAEKAFGNYGVYPRSI ClostridiumNEVGNAIVMMARGENEKCLVVVGEDSRLQELKGNQTE stercorariumENGLKVKVCPLSNENCYVIRKIFPYTNPQPHKGKNITIGLGDRLGLASPGHIRLIRDLDVFPVLAQQSIRELNLTGRTYEDVISAAAWAVFQEGYTKGYGADGDHLKTAEEVKMSLNVGMTMITLDCSEHIDNSAAHAGLSELREKYSRFTEEERERWERKYLNRDVKIGNYSFHISEEDLIRMACVYGGAIRHTLDIYHNIIAKCGRPIDFEMSIDETLTPTSPASHYFVAQELIDGGVEITSLAPRFCGEFQKGIDYIGDLKQFTDEFAVHAAIADHFGYKISVHSGSDKFKVFPVVGEKTNGRYHLKTAGTNWLEAVRVIARHKPDLYRRMHAFALEHLEDAKKYYHIGAKVENIPALETLADSELPELMNRDDSRQVMHITYGHILQAKDENGNPLFKDELYKVLYEYEEEYANALKKHIGRHLEGLGLL 329 pA06250MPSQLPEPLPVPPEARAHPSFRLHEGAALWLAGARLAVL TrueperaAPPEHPALTRFRGEVQHVGDHRLLRAERRAENAAALRAL radiovictrixLPDLQPRPLGLVTSAGFGDRLGVATPGHVRAAQRYGAGVAPVFAQQSIREMTRTGRTPQEVLDDATWGAFAAGWRGALGADADHQKTVADLERCAAAGFTLFTVDPSDHVDDSAHGAPASDLEAKVAALPWRELETTRADFERYAGRRLELGDRELVLAREAVLRAGAKYARAVLHVATLYRHLEGKGAPFELEVSVDETATPTSHAEHAVVALELRRLGVRWVGLAPRFVGRFEKGVDYRGDLGELKADLAGHAALARSLGPYKLSLHSGSDKFSVYPLIAEATGGMVHLKTAGTSYLEALRVAAQVAPGLFREILTLGRERFAVDKQSYHISAALARVSEADTLTDDELPRLLDDDDARQVLHVTFGSALDRYRAPLLRVLEAHDE AYQAGLAAHFAKHLTPFAEVAP 330pA06251 MLKLLNESLKPLSIFIYSESLRKINDDLYIFVAKIKDLKKIGIV DictyoglomusKQNQILYFSSPYFSEDKKIEGTNFLVNLYPLNFENYQKLKEI turgidumIPISPKVCDKKISFGTGDRLGLITSAQLSALKEYDLFPILAQQSPRELIKTKRDFKDVLLKSAMGVLETGYTGKYGADADHIKDEKYLMEAIDAGYTMYTLDISDFIEKIKDLSEKALKEKYEKVSSFSKKIIDKYAGKRVKISDEEYFELSYNELCKSAIVYEKALSFVEMVYEILKSKLSEFDIEVSIDEGERDTTPEDHFFVAQFLHDKGIDFKSLAPKFPGEFQKGIDYIGDIKEFERALKKHYALTKALEGYRLSLHSGSDKFSIYKIFYKITEGNFHIKTSGTSWLEAVKVIAKFFPDLFVELYQIALENLEESKKAYKVNITKEEFPKEIKEDYMEFLHKDNVRQLFHISYGVLLDEKRKEIYDLLN QKEKEHYQYVSENIKKHLKNLFEEE 331pA06252 MNDAVYALGRSSRNGTLQLIVRGNSTGFHGEQQGDALI CaldilineaCPLDAENARTLQERLPWLRPQPLGNRLSFGFGDRIGLAT aerophilaPGHVDALRSADPTGRIAPIFAQQSVRENQRLNRTPQEVMTAAVWSLFAENWRLPWGADADHVKEPEHLAPYVAAGYTFYTIDPSDHVDNAAHTDDLAVLRSKCEALPWDILETTYLSLCENYCGRTIVAEKTTLHFDKETLLRALAKYGRALAHTVRIAAALRTALGGTSFDLEMSVDETDTPTSAHEHFFIANELLRRNIPLVSLAPRFVGKFQKGVDYMGNLAEFEAELIRHVAVMRHFHCYKLSVHTGSDKFSIYPILARCAGENVHIKTAGTSYLEALRVAALRAPDLFRQMLETGRTCYEKDKKTYFLDCRPERVPPAATLDDADLPNLLDQFDARQLLHVTFGSILTTHGAALRNLLATYPNDYRSALRDHFARHIQPFVQA 332 pA06253MMLSPEALAEGLRLYGLHLIVGSIRELPDGGAIFAARQGS ThermoflexusERRIGWIGETSPFPAPDPRMSMRVQEHLVWIHPWTWA hugenholtziiHYRILRERLPALSPTRCDRPASFGAGDRLGMATAAQIAALERYPVFPVLAQQSPRELARTGRDFRSVLLDAAWGVFASGFAGPFGADADHLQDDEQLRAAAEAGYSLYTFDLRRALARGPRPWEALSPLARSVVAELADRRVEAPQGPRTLEESALRAAACRYEPALEEVVRGAEILRDQGIDADLEVSVDETEEETTPEAHAFIAVYLQRRGVALWSLAPRFPGVFEKAVDYEGEVERFAQAAALHTAVARTFGGHRLSLHSGSEKFRILPVFREATGGRFHVKTSGTTWLQAVRVVARAVPALFAELYAIARAHLEESRRDYPIALQPEALPPALPDDPEAALADRAVRQLFHISYGVLLRERGPAIRALLEAHEAEHFTAVRENLERHLEALL K 333 pA06254MIGNVLSTLEENGFKVYPDSLRKLGENIYIFVVKRQNEKM ThermoanaerobacteriumVGILSSSDVKLNGAYFSEDKNVSDKLRLNIYPFTFENYVTL thermosaccharolyticumNGKFHIGPTVCRGNSSFGTGDRLGLVTAAQLTALKKYDVFPILAQQSPRELIKTNRDFKDVLLKVVLGVLETGYIGHFGADADHIKDEYYLLEGINAGYTMYTLDLSEQLIDISSLNPSEMRNKAQELSQVSKDIIKDFSGKKLDIISDSGYVVSEEELYKSAVAYENAMKFVDKVNNILKEKLSDFDMEISIDEGGKVTTLEDHLYVAEYLHRNGIDFFSIAPKFPGEFEKAVDYIGDLDEFLLELKKHYQLSRMIGGYKISLHSGSDKFSIYRIFSDITEKNFHIKTSGTSWLQAINLIYNYDKEFYRELYKIALENLEESKKSYKVLIKREDFCKEPELNNPKFILKPEIKQLFHISFGVLLNLKRKEIVDFLNKYEEEHYKMVSKNIDNHLKEIFYKN 334 pA06255MFTILPKKGISLGLGDRIGIATPGHIKVAKKYNFFPVFAQQ Petrotoga mobilisSIRELNFTGRTFTDVRKDVLNALVEENYVGNSGFDGDHLKSDEEIQYALDSGITMLTLDCSEHMNKDSSIKDQIFEQFYNKSFFVNDMPIEYSDKNELNKIVSIYASVIERVIDVWNKFPKVNKKEVTFEVSVDETDVPTDEKTHFLISKYIYDEGVKIDTLAPRFPGEFQKAIDYIGNIQEFKKSLIKQDKIAKYFGYRLSIHSGSDKFSIYPIIGEVTQGNYHLKTSGTSYLEAIKVVAQKDPEFFKKIWQTCLDKREEMDKYYHLSCDPFSVPKDLSPTEYLQNPDARQTLHVSYMFVLNPQYDFREKFFEILTKYQNEY HENVANHIEKHVKELKIEEKS 335pA06256 MATPGSLSFPRYSIGTGDRFGHEAEAQLRAVIEAGRLGR SpirochaetaALGIVWNKSYREHTIIGSRPEDVRRMADRAVSSLGWEG thermophilaPYFVDADHITTKTVDLFLDSADFFTIDVAEAIGKGEVSPQEEEDLLASLGDLLNRELAIPGLSSPLAISEETARGTIRAYWPAVREAARIYRRIEQGASRPFVVEVSMDETDEPQRPPELLLILAMIRKAGIPARTIAPKFSGAFYKGVDYVGDPHTFAREFEDDLCVVRYAREQFALPEGLKLSVHSGSDKFSLYPLVREILSRHPQEGVHLKTAGTTWLEEVAGLAEAGGEALALAKEIALTCYSMIEELCAPYAAVIDIDPERLPSPGEIEEWSSGRFVEALEHDPSNPSYNRDFRQLIHVGYKVAAQMGERFHQALEAH REVIAARVTRNLLERHIIPLFPGDIP 336pA06257 MLHVYLGKIPRPGFGIRIPEVVAPPLLSAFKSLGMTGSLM ThermofilumLSFNRETAPAEYIESSDPRLFYFGHTGTSIGGFIRSVKEYSK pendensALSVPVEVEADHVSILGSVERALKKIAGVPVEEPLSEEEVSWSIGYVERELREAAEAGGVDFVTIDTCELIDYSYDKVGAEEVAAAYEEVFDGDERRALEERYEGVHYFLGGDRVVAVRLSREDVARLAVKYRRSLDYAERIYRAAREAMGVELGFEVAFDETPGVSEAREVFFYLSELLRRGLRVDFIAPNVGFRKREDYSGDLHALYERLRNLHAVVSSMNAYLSIHSGSGSHPYSDKGFGVWGVVGRATGGAVKYKMSGVLVQLLLEVMASYPPGSETRRLYEEIYSEVVEHLRWVVKAKASLYSPELETLLKRYEAAQDRFDPRADVFRHYFYVFQALRDEGGARRLRERLVEHYRENPGLRERYEKELRGLVERLASQLGYAGNAYRYRVV YA 337 pA06258MVTVLQTLLQRPRPLAEIDRAALARFLTDLIRQQVYPASL RhodothermusEPTSEGVFFLARDGREKRLGILSEAGLHDFEGARHQLSLD marinusGRTLIFQSCPLTAANARALRQHLAWTAPRPLGLRASVGCGDRLGLATPGHVRAVRKHKLAPVFAQQSIREMTRTGRTPQQVLDEAMWGVFQEGWRQGYGADADHLKTEEDADRCIEAGFTFFTIDPSAYVDNEVDTADAATLEAKVAALPWDALETTLADLRRAYLGQHFQVGPYELSFEERTLLQALAKYGGAIAHTARVYRHIAGRMGNRPFELEMSVDETEVPTSPAEHFFVARELQRLGVRWISLAPRFVGRLEKGVDYIGDLEEFEAHLKLHVAIARTLGPYKLSLHSGSDKFALYPLFARHAGELFHLKTAGTSYLEALRAVAELDPPLFREILDFARDRYETDRATYHVSALLERVPKASDVPDDALPALLEQFDTRQVLHVTFGSVLTATDADGRPRFRDRLLAVLQENEETYYRLLEAHFDR HLAPFDAK 338 pA06261MLNLLEELLKPFSIFVYPQSLRKINEELYIFVAKINNTKNIGII DictyoglomusKQNQSIYFSNPYFSEDKKIEKTGFSVNIYPLNFENYQKLKEI thermophilumIPINPKTCNKKISFGTGDRLGLVTSAQLSVLKDYNLFPILAQQSPRELIKTKRDFKDVLLKSVIGVLEIGYTDSYGADADHIKDEKYLMEAIDAGYTMYTLDISDFIERIENLTSKEIREKYEKISSFSKKIIEKYAGKKIKISNEEYFELSYDELCKSAIVYEKALSFVEMVYEILRSKLLEFDIEVSIDEGERDTTPEDHFFVVQFLHEKGIDFKSLAPKFPGEFQKGIDYIGDIKKFENELKKQYALTKALEGYRLSLHSGSDKFSIYKSFYKITEGNFHIKTSGTSWLEAVKTIARYSPDLFLELYHIALENLEESKKAYKVSITKEEFPKEIKEDYIEFLKKPNVRQLFHISYGVLLDEKREEIYEILNKNEKE HYQYVSENIRKHLKNLFEEE 339pA06262 MKEELSDYLLKNSFLLYPDSFRRLREDVYIFVAKKDSDKKI ThermoanaerobacterGLLTNGNFKLSSPHFAEDKYVEELGFYINLYPLTYENYLILK siderophilusDNFGISPVTCKEKASFGTGDRLGLATPAHIKALKNYNVFPVLAQQSPRELVKTHRDFKDVFLKVILGVLEAGYAGGYGADADHIKDEKYLIEAIDAGYTMYTLDLSDLLVKISDMPKSQLKEKAQSLSSQSREIIDRFKGKKFSISTDEDFAVSEDELYKSALTYEKAMKFVEKVYGILKDRLQHFDLEISIDEGEKDTTVEDHIFVAEYLHRKGIDFWSLAPKFPGEFQKAIDYKGDIKKFTSGLKKHYFLSKKLGGYKLSLHSGSDKFSIYKIFNEITEGNFHIKTSGTSWLQAINIIFERDKDLFNDLYKIALDNLEESKKAYKVLIDRDDFPQTIQTEDSQILLKPEIKQLFHISYGVLLDERRKEIYEVLNKYEEEHYEFVSKNIENHLKEIFNI 340 pA06263MVEKSILEKLTDFLLNHSFVLYPNSLRKLKEDTYIFVAKKD ThermoanaerobacterADKKIGILTKENFKLTSPYFVEDKNVKEIDFYLNLYPLSFEN mathraniiYLILKNFGISPTPCRQKSSFGTGDRLGLVTPAHIVALKEYPVFPVLAQQSPRELEKTHRDFKDALLKVILGVLEAGYTGEFGADADHIKDEKYLLRAIEAGYTMYTLDVSELLTKILDISSNQVMQISPQSKEIIEAFKGKKISISEEEYTIREDELYKSALIYEKAMNFVEKVYSILKEKVKDFDLEISIDEGEKDTTVEDHIFVAEYLHKKGIDFWSLAPKFPGEFQKAIDYKGDINKFAVELKKHYAISQQLGGYKLSLHSGSDKFSIYEIFSEVTQHSFHIKTSGTSWLQAVNLIFEKNKKLFYELYKIALNNLEESKKAYKVLIDKDDFAEEPNLENVQILSQPEIKQLFHISYGVLLDEKKEEIY DVLDKYEEEHYQFVSANIKNHLGKIFNN341 pA06264 MVEKGILEKLTDFLLNHSFVLYPNSLRKLKEDTYIFVAKKDThermoanaerobacter  ADKKIGILTKENFKLSSPYFVEDKNVKEIDFYLNLYPLSFEN italicusYLILKNFGISPTPCRQKSSFGTGDRLGLVTPAHIVALKEYPVFPVLVQQSPRELEKTRRDFKDALLKVILGVLEAGYTGEFGADADHIKDEKYLLRAIEAGYTMYTLDVSELLTKISDISSNQVMQISPQSKEIIEAFKGKKISISEEEYTIREDELYKSALIYEKAMNFVEKVYSILKEKVKDFDLEISIDEGEKDTTVEDHIFVAEYLHKKGIDFWSLAPKFPGEFQKAIDYKGDINKFAVELKKHYAISQQFGGYKLSLHSGSDKFSIYEIFSEVTQHSFHIKTSGTSWLQAVNLIFEKDKKLFYELYKIALNNLEESKKAYKVLIDKDDFAEEPNLENAQILSQPEIKQLFHISYGVLLDEKKEEIY DVLDKYEEEHYQFVSANIKNHLEKIFNK342 pA06265 MIGNVLSTLEENGFKVYPDSLRKLGENIYIFVVKRQNEKMThermoanaerobacterium VGILSSSDVKLNGAYFSEDKNVSDKLRLNIYPFTFENYVTLthermosaccharolyticum NGKFHIGPTVCRGNSSFGTGDRLGLVTAAQLTALKKYDVFPILAQQSPRELIKTNRDFKDVLLKVVLGVLETGYIGHFGADADHIKDEYYLLEGINAGYTMYTLDLSEQLIDISSLNPSEMRNKAQELSQVSKDIIKDFSGKKLDIISDSGYVVSEEELYKSAVAYENSMKFVDKVNNILKEKLSDFDMEISIDEGGKVTTLEDHLYVAEYLHRSGIDFFSIAPKFPGEFEKAVDYIGDLDEFSLELKKHYQLSRMIGGYKISLHSGSDKFSIYRIFSDITEKNFHIKTSGTSWLQAINLIYNYDKEFYRELYKIALENLEESKKSYKVLIKREDFCKEPELNNPNFILKPEIKQLFHISFGVLLDLKRKEMVDFLNKYEEEHYKMVSKNIDNHLKEIFYKN 343 pA06266MIGNVLSTLEENGFKVYPDSLRKLGENIYIFVVKRQNEKM ThermoanaerobacteriumVGILSSSDVKLNGAYFSEDKNVSDKLRLNIYPFTFENYVTL thermosaccharolyticumNGKFHIGPTVCRGNSSFGTGDRLGLVTAAQLTALKKYDVFPILAQQSPRELIKTNRDFKDVLLKVVLGVLETGYIGHFGADADHIKDEYYLLEGINAGYTMYTLDLSEQLIDISSLNASEMRNKAQELSQVSKDIIKDFSGKKLDIISDSGYVVSEEELYKSAVAYENAMKFVDKVNNILKEKLSDFDMEISIDEGGKVTTLEDHLYVAEYLHRNGIDFFSIAPKFPGEFEKAVDYIGDLDEFLLELKKHYQLSRMIGGYKISLHSGSDKFSIYRIFSDITEKNFHIKTSGTSWLQAINLIYNYDKEFYRELYKIALENLEESKKSYKVLIKREDFCKEPELNNPKFILKPEIKQLFHISFGVLLNLKRKEIVDFLNKYEEEHYKMVSKNIDNHLKEIFYKN 344 pA06267MIGNVLSTLEENGFKVYPDSLMKLGENIYIFVVKRQNEK ThermoanaerobacteriumMVGILSSSDVKLNGAYFSEDKNVSDKLRLNIYPFTFENYV thermosaccharolyticumTLNGKFHIGPTVCRGNSSFGTGDRLGLVTAAQLTALKKYDVFPILAQQSPRELIKTNRDFKDVLLKVVLGVLETGYIGHFGADADHIKDEYYLLEGINAGYTMYTLDLSEQLIDISSLNPSEMRNKAQELSQVSKDIIKDFSGKKLDIISDSGYVVSEEELYKSAVAYENAMKFVDKVNNILKEKLSDFDMEISIDEGGKVTTLEDHLYVAEYLHRNGIDFFSIAPKFPGEFEKAVDYIGDLDEFLLELKKHYQLSRMIGGYKISLHSGSDKFSIYRIFSDITEKNFHIKTSGTSWLQAINLIYDYDKEFYRELYKIALENLEESKKSYKVLIKKEDFGKEPELNNPKFILKPEIKQLFHISFGVLLDLKRKEIVDFLNKYEEEHYKMVSKNIDNHLKEIFYKN 345 pA06268MIGNVLSTLEENGFKVYPDSLRKLGENIYIFVVKRQNEKM ThermoanaerobacteriumVGILSSSDVKLNGAYFSEDKNVSDKLRLNIYPFTFENYVTL thermosaccharolyticumNGKFHIGPTVCRGNSSFGTGDRLGLVTAAQLTALKKYDVFPILAQQSPRELIKTNRDFKDVLLKVVLGVLETGYIGHFGADADHIKDEYNLLEGINAGYTMYTLDLSEQLIDISSLNASEMRNKAQELSQVSKDIIKDFSGKKLDIISDSGYVVSEEELYKSAVAYENAMKFVDKVNNILKEKLSDFDMEISIDEGGKVTTLEDHLYVAEYLHRNGIDFFSIAPKFPGEFEKAVDYIGDLDEFLLELKKHYQLSRMIGGYKISLHSGSDKFSIYRIFSDITEKNFHIKTSGTSWLQAVNLIYKFDKEFYRKLYKIALSNLEESKKSYKVLIKKDDFKDEPELDNPEFTLRPEIKQLFHISFGVLLDLKGKEIKDMLNDYEEEHYKMVSDNIENHLKEIYYEK 346 pA06269MVGNVSSVLKESGFQIYPDSLRKLGENTYIFVVKKQKEK ThermoanaerobacteriumMIGILSNDELKLKEPYFSENKKISDNLQFNVYSFTFDNYVT xylanolyticumLNGRFHIGPTICRENASFGTGDRLGLATAAQLDALKKFNVFPILAQQSPRELVKTNRDFKDVLLKVVLGVLETGYIGHYGADADHIKDEKYLLEGIDAGYTMYTLDLSEQLFDVSGATSLEIKEKAKTLSDVSRKIVEDFSGKSLNVGFGGHLVSEDELLKSAVAYEAAMKFVEKVNDILKEKLNDFDLEISIDEGGKVTTLEDHLFVAEYLHRNGIDFFSIAPKFPGEFEKAIDYVGDVNEFERELKKHYDLTKLIGGYKLSLHSGSDKFSIYKIFSQTTEKNFHIKTSGTSWLQAVNLIYKSDKEFYRELYKIALSNLEESKKSYKVLIKKDDFKDEPELDNSEFIIRPEIKQLFHISFGVLLDLKGKEIKDMLYDYEEEHYKMVSDNIENHLKEIFYEK 347 pA06270MLTILPNKGISLGLGDRIGIATHGHIKVAKKYNFFPVFAQ Petrotoga mobilisQSIRELNFTGRTFSDVRKDVLNALIEENYVGNSGFDGDHLKSDEEIQYALDSGITMLTLDCSEHMNNKDSSIKERIFDQFYNKSFFVNDMPIEYSNKNELNKIVSIYAGVIERVIDVWNKFPKVNKKEVTFEVSVDETDVPTDEKTHFLISKYIYDEGVKIDTLAPRFPGEFQKAIDYIGNLQEFKKSLIKQDKIAKYFGYRLSIHSGSDKFSIYPIIGEVTQGNYHLKTSGTSYLEAIKIVAQKDPEFFKKIWKTCLDKREEMDKYYHLSCDPFSVPKNLSPTEYLQNPDARQTLHVSYMFVLNPQYDFREKFFEILTKYQNE YHQNVANHIEKHVKELKVEEKS 348pA06271 MVGNVSAVLKENGFKIYPDSLRKLGESTYIFVVKKQKEK ThermoanaerobacteriumMIGILSNDELNLKEPYFSENKKISDNLQFNVYPFTFDNYVT saccharolyticumLNGRFHIGPTVCRENASFGTGDRLGMATAAQLGALKKFDVFPVLAQQSPRELVKTNRDFKDVLLKVVLGVLETGYIGHYGADADHIKDEKYLLEGIDAGYTMYTLDLSEQLFDISGATPSAIKEKAEALSDVSKKIVEDFSGQSLNVGLEGHLVSEDELLKSAIAYEGAMKFVEKVNDILKEKLNDFDLEISIDEGGKVTTLEDHLFVAEYLHRNGIDFFSIAPKFPGEFEKAIDYVGDVDEFKKALKKHYDLTKLIGGYKLSLHSGSDKFSIYKIFSQTTERNFHIKTSGTSWLQAVNLIYKSDREFYRELYKIALSNLEESKKSYKVLIKKDDFKDEPELDNPEFIVRPEIKQLFHISFGVLLDLKGKEIKDMLYEHEEEHYKMVSNNIENHLKEIYYEK 349 pA06272MFTILPKRGISLGLGDRIGIATTGHIKVAKKYNFFPVFAQQ Petrotoga mobilisSIRELNFTGRTFIDVRKDALNALVEENYVGNSGFDGDHLKSDEEIQYALDSGITMLTLDCSEHMNKDSSIKDQIFEQFYNKSFFVNDMPIEYSDKNELNKIVSIYASVIERVIDVWNKFPKVNKKEVTFEVSVDETDVPTDEKTHFLISKYIYDEGVKIDTLAPRFPGEFQKGIDYIGNIQEFKKSLIKQDKIAKYFGYRLSIHSGSDKFSIYPIIGEVTQGNYHLKTSGTSYLEAIKVVAQKDPEFFKKIWQTCLDKREEMDKYYHLSCDPFSVPKDLSPTEYLKNPDARQTLHVSYMFVLNPQYDFREKFIEILTKYQNEYH QNAANHIEKHVKELKVEEKIHKQKN 350pA06273 MTTPGSLSLPRYSIGTGDRFGHEAEAQLRAVIEAERLGM SpirochaetaALGIVWNKSYREHTIIGSRPEDVRRMADKAVSALEWEGP thermophilaYFVDADHITTKTVELFLDSSDFFTIDVAEAIGQEEISPQEEEDLLSSLDDLLNRELAIPGLSNPLTISEETARETIRAYWPAVREAARIHQRIEKGTSRPFVVEVSMDETADPQRPPELLLILAMIRKAGIPARTIAPKFSGSFYKGVDYVGDPEVFAREFEDDLCVVRYAREAFRLPEGLKLSVHSGSDKFSLYPLIKDILSRHPQEGVHLKTAGTTWLEEVTGLAESGGEALALAKEIALTCYSMIEELCAPYAAVIDINPDRLPSPGEIEDWSSGRFVEALEHDPSNPSYNRDFRQLIHVGYKVAAQMGERFHEALEAHRE VIAARVTRNLLERHIIPLFPGGAA 351pA06274 MRAKELFGEDVSIEIALDESPSETQLKELFFYINELLYKGLR IgnisphaeraFEFIAPNIGFRKREDYRGDLQELYNRVRKLHTIASNNGVY aggregansLSIHSGSGAHPYSDKGVGVWSTIGRATDGLVKYKMSGVLIQLLLEVMSRFPKGSTVRRVYEEIYDAVLDHLKKDISRGRGLASETLRKMIEDYEEHSNKYDVRADVFRHYFFVFQCIRDDSGVRYLRNRVIELFNEVKELRDRYREEVANLITREAEALGY INSVIRYRKYEYS 352 pA04536MVLKVFKDHFGRGYEVYEKSYREKDSLSFFLTKEEEGKILV ThermotogaVAGEKAPEGLSFFKKQRAEGVSFFFCERNHENLEVLRKYF maritimaPDLKPVRAGLRASFGTGDRLGITTPAHVRALKDSGLFPIFAQQSVRENERTGRTWRDVLDDATWGVFQEGYSEGFGADADHVKRPEDLVSAAREGFTMFTIDPSDHVRNLSKLTEKERNEKFEEILRKERIDRIYLGKKYSVLGEKIEFDEKNLRDAALVYYDAIAHVDMMYQILKDETPDFDFEVSVDETETPTSPLFHIFVVEELRRRGVEFTNLALRFIGEWEKGIDYKGDLAQFEREIKMHAEIARMFEGYKISLHSGSDKFSVYPAFASATGGLFHVKTAGTSYLEAVKVISMVNPELFREIYRCTLDHFEEDRKSYHISADLSKVREVEKVKDEDLPGLFEDINVRQLIHVTYGSVLKDASLKERLFKTLEQNEELFYETVAKHIKRHVDLLEG 353 pA04539MINKVAEYLSGEGFYFYEKSFRKLSEDIYIFVVKKANEKSIG CaldanaerobacterLLTQGDFTLSSPHFTERKYLKETGYYLNLYPLTYENFVILKD subterraneusKFNIAPAPCDKKASFGMGDRLGLVTAAHIRAVENYDVFPVLAQQSPRELMKTHRSFKEAILKAILGVLEEGYTGKFGADADHIKDENYLMEAIDAGYTMYTLDLSDMLVKLSDYTESQLKEKAEKLNITSKRIIERFKGKKFVMPTKEAFTVSEEELYKSALTYEKAMDFVEKVYGILKDKVKNFDLEISIDEGDKDTTVEDHIFVAEYLHEKGIDFWSLAPKFPGEFQKAIDYIGDVDKFAVELKKHQFLSREFGGYKLSLHSGSDKFSIYKVFSEITEGEFHIKTSGTSWFQAVNLIFEKDKELFKELYQIALYNLEESKKAYKVLIDKKDFPENINLEDSQIVSKPEIKQLFHISYGVLLEERKKQIFEVLNKYEEEHYEFVRKNIENHFKEIFSK 354 pA07082MKENKVMNSFSAIYGNRYKVYEKSLKRKEKEFFFVIKDFQ Mesotoga inferaRKYLVAAGPSYRIRKRDFQPDEEGVADNEGEFLFQICRLTHHNLTQLQSIFNYLQPSTTKMKPSFGTGDRLGIATPAHIQAFEDKNIFPILAQQSVREMERTESNWQKVLDNAIWGCFEAGYEGKFGADADHVKDLKNLKEAIDCGFTFYTIDPSDHIDANILKLDKDELRNKYQQLPEKDALENSYLNKEYQIGSQKLTFTQDILIEIVLTYLEAIKHVEKCYKFLKDSHKGDFELEVSVDETPTPTSPLAHLWIASELQRRGVDFQNLAPHFIGDWEKGIDYIGNIDTFKEEFKLHCQIASQMGGYKLSLHSGSDKFSVYPIFAEETNGYFHVKTAGTSWLEAVKAIVVCDPALYREMYEFALKCFEKDSFSYLLSTDLQKIPNIKELQDKELIQLFSNNNARQLIHITYGSILREKDSQNRYKFRDRIYKVLFENEDIH YENVSKHIRHHLGLLSV 355 pA07083MQSREELKRAILSEFGDYNIYSESIYRAGGCVLFLAKDMG ThermosinusQKLLVVVEEETGSAFDRFVGPQVYHPSGKRVKEAPLQPV carboxydivoransNAGIVRELLPFTAPVALGATGLSLGLGDRLGVASPGHLRLIKKTGVRPVLAQQSVRELTLTNRTYSDVLDAATWAVLQEGYEGGFGADGDHLKTAEEIKGALDLGFTMITLDASAHIDNTVGQKAAKQVAELYHTLPADYTADMEEHYLGKAFIVGGMAITFDTETLQRLVLTYGKALAFMGYIYHTLIVNAGREVDFEISIDETATPTTPAAHYFVASELGRMGVKFTSLAPRFCGEFQKGIDYIGDLYQFEDEFKRHAAIADHFGYRLSIHSGSDKFSVFPIIGQYTRGRVHIKTAGTNWLEALRVVARINPALFREIYAFAGEVFGEAKKYYHVTTDLTRLPDVAAMADDGLPTVLDHNDARQMLHITYGLVLTAANADGSYRFKDALYEL LFDHEDEYYAALERHIGRHLEKLTENLKG356 pA07084 MSWKDFAEELVGTSKEAVMKVAEYAEDYRIYPRSIIKKDK HalanaerobiumSFYFLAKIDQKKKLVILNKSKNFELFQGRIEELAGFKAKIGP congolenseLSHYNAEILREVFPFTAPSALGNKKPSIGLGDRLGIATPGHIEAVKESAAMPVFAQQSVRELNLTGRTFKSVLDDVSWAVFQEGYQAGFAADADHLKEKPDIKEALDLGYTMLTLDCTDYINDDLDQMSESEIENAYAEVPDYLREGLENQYLNKTFVLNSGYQLEYNQDNFKEIVLIYYKMLDFAKEIQHLIKTSARNVDFEISIDETSTPTTPEAHFFVANELKRNNIEVNSLAPRFVGEFQKGIDYIGDLEQFEKEFKVHADIADRFGYKLSIHSGSDKFSVFPIIGRHTQGRVHVKTAGTNWLEAIRVVAENNPSLYRDIHAYALKKFEAAKEYYHVTTDLDKVPELARMSDQELGELLEINEVRQLLHITYGFILQDKKDGRYIFRDKLYKFWDE YDKEYRRALERHIGRHLNKLGFYKN 357pA07085 MSWKDFAEELVGTSKEAVMKVAEYAEDYRIYPRSIIKKDK HalanaerobiumSFYFLAKIDQKKKLVILNKSKNFELFQGRIEELAGFKAKIGP congolenseLSHYNAEILRKVFPFTAPSALGNKKPSIGLGDRLGIATPGHIEAVKESAAMPVFAQQSVRELNLTGRTFKSVLDDVSWAVFQEGYQAGFAADADHLKEKPDIKEALDLGYTMLTLDCTDYINDDLDQMSESEVENAYAEVPDYLREGLENQYLNKTFVLNSGYQLEYNQDNFKEIVLIYYKMLDFAKEIQHLIKTSARNVDFEISIDETSTPTTPEAHFFVANELKRNNIEVNSLAPRFVGEFQKGIDYIGDLEQFEKEFKVHADIADRFGYKLSIHSGSDKFSVFPIIGRHTQGRVHVKTAGTNWLEAIRVVAENNPSLYRDIHAYALKKFEAAKEYYHVTTDLDKVPELARMSDQELGELLEINEVRQLLHITYGFILQDKKDGRYIFRDKLYKFWDE YDKEYRRALERHIGRHLNKLGFYKN 358pA07086 MSWKDFAEELVGTSKDAVKKVAEYAEDYRIYPRSIIKKEK HalanaerobiumSFYFLAKIDQKKKLVILNKSKNFDIFQGKTEKLAGFKAKIAP saccharolyticumLSHYNAEIIREVFPFTAPSKIGNQTASIGLGDRLGIATPGHIEAVKKSNAMPVFAQQSVKELKLTGRSFKSVLDDVSWAVFQEGYQNGFGADADRLKEKPEIKEALDLGYTMLTLDCTDYINDNFDQTAESDIESAYAEVPDYLRDGLESKYLNKTFVLNSGYQLEYNKVNFKKIVLSYYQILDFVKEIQHLIKRSARDVDLEISIADSLNSTSPEAHFFVANEFKRNNIEVNSLALNFVGEFQKGIDYIGDLEKFEKDFEIHADIADRFGYKLSIHSGSDKFSIFPIIGRQTEGRAHIKTAGTNWLEAIRVVAENDPSLYREIHSYALKKFEEAKEFYQVNTDLSKVPELAVMSDQELGELLEIDAVRQLLHITYGFILQDKKDGRYIFRDRLYKLLDEYDKDY RSGLERHIGRHLNKLGFYKN 359pA07087 MVKHFESVLEELSQRKVPTSTEEVTVYTPSFEEHAGSQVV GracilibacillusMVKSGTEKMIVAAGAGELFEALSGEDIGKGKVCPLTHEN halophilusRLVLNQFFSYTAPQAFGTDIATMGLGDRLGIASPGHIDTVKERNVKPILAQQSIRELTLLNRTMTDILDAAAFAVFQEGYKDGYGADADHIKLESDIEHALQLGFSFLTLDCSEQIRNDVESQTSDEIQNEFASLSDEKRAYFSNYYLDQTFNVHERQISFDQANLAKNVLVYGEAIDFMEHVYHTYLQSLDRDVDFEISIDETETVTSPEAHFFVAEELRRRGVKVESLAPRFCGEFQKGIDYIGDMDQFEKELKEHADIAKHFGYKLSIHSGSDKFSVFPIIGKYTDGLLHIKTAGTNWLEAVRVVAQENPDLYRRMHVYAEEHFEETLKYYHVTPDLDSVTPLKEQPDDQLPEYMNHDAARQLFHVTYGILLTAKDDAGNDLFRDEFFDTLLNK EDAYRQALAHHIGRHLDLLGLSKKVGIE 360pA07088 MINKVAEYLSREGFHFYEKSFRKFSEDIYIFVVKKANEKSIG CaldanaerobacterLLTQRDFTLSSPYFTERKYLKEIGYYLNLYPLTYENFVILKDK subterraneusFSIAPSPCNKKVSFGMGDRLGLVTAAHIRAVQNYDVFPVLAQQSPRELMKTHRSFREAILKAILGVLEEGYTGKFGADADHIKDENYLMEAIDAGYTMYTLDLSDMLVKLSDYTESQLKEKAEKLNITSKRIIEKFKGKKFVMPTEEAFTVSEEELYKSALTYEKAMDFVEKVYGILKDKVKNFDLEISIDEGDKDTTVEDHIFVAEYLHEKGIDFWSLAPKFPGEFQKAIDYIGDVDKFAVELKKHQFLSREFGGYKLSLHSGSDKFSIYKVFSEITEREFHIKTSGTSWLQAVNLIFEKDKKLFKELYQIALYNLEESKKAYKVLIDKKDFPENINLEDSQIVSKPEIKQLFHISYGVLLEERKKQIFEVLNKYEEEHYEFVRKNIENHFKEIFSK 361 pA07089MLPYLVARPLGLRKSAGCGDRLGLATPGHIRALRATFGR LitorilineaDEDAAMAPIFAQQSIRENARTGRTPQEVMDDAMWGV aerophilaFQEGWRAGFGADADHLKTLADVDICAAAGYTFYTVDPGDHVDDEANTAAFPALEARVDALPWDVLDSSPADLAARLADRPIDLGTLKVTLDRETLWRAAAKYGRAVAHTVTMYRHLAGAMGERPFELEMSVDETATVTSLAEHVYIAAELQRLGVRCVSLAPRYVGTFEKGVDYIGDLDAFEQSIAQHMAVSRTFGPYKLSLHSGSDKFSIYPIASRVAGDLVHLKTAGTSYLEALRAIAAMAPDLFRQIVAFARERYPTDRASYHVSAELEKMPDIAGWPDDRLPELLNDFHAREILHVTFGSVLNHPPFR EPFFTALRTHEETYSEMLERHFCRHFAPFAG362 pA07090 MINKVAEYLSREGFYFYEKSFRKFSEDIYIFVVKKANEKSIG CaldanaerobacterLLTQGDFTLSSPYFTERKYLKEIGYYLNLYPLTYENFVILKDK subterraneusFSIAPSPCNKKVSFGMGDRLGLVTAAHIRAVENYDVFPVLAQQSPRELMKTHRSFKEAILKAILGVLEEGYTGKFGADADHIKDENYLMEAIDAGYTMYTLDLSDMLVKLSDYTESQLKEKAEKLNITSKRIIERFKGKKFVMPTKEAFTVSEEELYKSALTYEKAMDFVEKVYGILKDKVKNFDLEISIDEGDKDTTVEDHIFVAEYLHEKGIDFWSLAPKFPGEFQKAIDYIGDVDKFAVELKKHQFLSREFGGYKLSLHSGSDKFSIYKVFSEITEGEFHIKTSGTSWLQAVNLIFEKDKELFKELYQIALYNLEESKKAYKVLIDKKDFPENINLEDSQIVSKPEIKQLFHISYGVLLEERKKQIFEVLNKYEEEHYEFVRKNIENHFKEIFSK 363 pA07091MFERKIEMINKVAEYLSREGFYFYEKSFRKFSEDIYIFVVKK CaldanaerobacterANEKSIGLLTQGDFTLSSPYFTERKYLKEIGYYLNLYPLTYE subterraneusNFVILKDKFSIAPSPCNKKVSFGMGDRLGLVTAAHIRAVENYDVFPVLAQQSPRELMKTHRSFKEAILKAILGVLEEGYTGKFGADADHIKDENYLMEAIDAGYTMYTLDLSDMLVKLSDYTESQLKEKAEKLNITSKRIIEKFKGKKFVMPTEEAFTVSEEELYKSALTYEKAMDFVEKVYGILKDKVKNFDLEISIDEGDKDTTVEDHIFVAEYLHEKGIDFWSLAPKFPGEFQKAIDYIGDVDKFAVELKKHQFLSREFGGYKLSLHSGSDKFSIYKVFSEITEGEFHIKTSGTSWLQAVNLIFEKDKKLFKELYQIALYNLEESKKAYKVLIDKKDFPENINLEDSQIVSKPEIKQLFHISYGVLLEERKKQIFEVLNKYEEEHYEFVRKNVENHFKEIFSK 364 pA07092MGMMDKDVLNQLSSLLSRHSFVLYPNSVRNLAEDIYVF CaldicoprobacterVAKGNADKKVGILSKGKALGFKAPFFAEDIKVEATGFSFN faecalisLYPLSFENYLILRDEFGIALVPCKNKASFGTGDRLGLATPAHLDAFKSYNMFPVLAQQSPRELEKTHRDFRDVLLKAVLGVLEAGYTGEFGADADHIKDERYLLEAADAGYTMYTLDVSEMLVKGDVSPDKADHLSQHSRDIIKDFSGKRISFEGGEYTVKEEELYRSAVIYEKAMNFVERVHGLLKERLKDFDLEVSIDEGDRDTTVEDHIFVAEYLHRRGIDFWSLAPKFPGEFEKAVDYRGDIDKFTVELNKHCAVARMLGGYRLSLHSGSDKFSVYRIFNDATQHNFHIKTSGTSWLQALNVIHEKDRQLFKELYNIALDNLEESKKAYKISIYRQDFEEGLDLDNLHVLQNPKVKQLLHISYGVLLDEKRQEIYEVLNQHEAEHYRYVSDNIKKHL ELLK 365 pA07093MKEELSNYLLKNSFLLYPDSFRRLKEDVYIFVAKKDSDKKI ThermoanaerobacterGFLTNGNFKLSSPHFLEDKYVEELGFYLNLYPLTYENYLILK uzonensisDNFGISPVTCKEKTSFGTGDRLGLVTPAHIKVLKNYDIFPVLAQQSPRELVKTNRDFKDVLLKAILGVLEAGYAGGFGADADHIKDEKYLMEAIDAGYTMYTLDLSDLLVKISDMPESQLKEKAQSLSSQSREIIDRYKGKKFSISTDEDFVVSEDELYKSALTYEKAMKFVEKVYGILKDRLQHFDLEISIDEGEKDTTVEDHIFVVEYLHRKGIDFWSLAPKFPGEFQKAIDYKGDIKKFTSELKKHYFLTKELGGYKLSLHSGSDKFSIYKIFNEITEGNFHIKTSGTSWLQAISLIFEKDKDLFNDLYKIALDNLEESKKAYKVLIDRDDFPQTIQTEDSQILSKPEIKQLFHISYGVLLDERRKEIYEVLNKYEEEHYEFVSKNIKNHLKEIFNI 366 pA07094MDLNGLLKDVEEILAKVDAGEKVESLSDAGVYVPSVQVD LactobacillusRRNVYFIYHTKDEKDHTVKTLVVYEENPTIGDFDALETLK ingluvieiGENSTLITAALTDHNNQALAKRFPWIKPTSRRNYKYTFGLGDRLGNASNAHLRLFKGTGIMPVLAQQSIRELTLMHRTNTDVLLSASWAVFEEGFTFGWGADGDHVKTEYEVDYAVKVGCSMITLDCTDVINNDAVTMSDEELDKTFNALDDDQKKYFNDTYLDKTFDLGNGNSVHFTKHDVEESVLTFYGAILFAADIYKKFVVPYNLDFEISMDETPYQTTNPNHFFFGNELHKRGIVPTTMAPRFYGEFQKAIDYIGDKDRFEREFVLHEAIAEYFGYKLSIHSGSDKLSVYEIIGRVAKNGWHVKTAGTNWLEALRVIAHKDPEFMVELYKYAYEHLDDVKDFYVFNAQTDGKAPKPENVTVDNVVDVLSDDDGRQVLHTMYGSLMNLKHNYHYVFRDKFWDILLKNQDLYDKYLNIHIAEHID LLQGKYKTKEEALAALEPKTDISKEY 367pA07095 MFTILPKKGISLGLGDRIGIATTGHIKVAKKYNFFPVFAQQ PetrotogaSIRELNFTGRTFTDVRKDVLNALIEENYVGNSGFDGDHLK mexicanaSDEEIQYALDSGITMLTLDCSEHMNKDSSIKDQIFEQFYNKSFFVNDMPIEYSDKNELNEIVSIYASVIERVIDVWNKFPKVNKKEVSFEVSVDETDVPTDEKTHFLISKYINDEGVKIDTLAPRFPGEFQKGIDYIGNVQEFKKSLIKQEKIAKYFGYRLSIHSGSDKFSIYPIIGEVTQGNYHLKTSGTSFLEAIKVVAQKDPEFFKKIWQTCLDKREEMDKYYHLSCNPFSVPKDLSPTEYLKNPDARQTLHVSYMFVLNPQYDFREKFFEILTKYQNEYH ENVANHIEKHVKELKIEETIHKQKK 368pA07096 MFSKLPKQGISLGLGDRVGLATPGHIKVAKRHEFFPVFA DefluviitogaQQSIRELNFTGRTFHDVKKDVENAVIKENYEGKSGFDGD tunisiensisHLKTDEEIKMAIDSGITMLTLDCSEYMGVVSKIKEKIYKGFYGKTFKVKDLDLEYSQEELEKILSIYSGVIERIIYIWNNFPKVKNKDVSFEVSIDETNIPTDEKTHFLLSKYLYDEGITIDTLAPRFPGEFQKGIDYIGNIKEFKNSLMKHHKIASYFGYRLSIHSGSDKFSIYPYVSQITQGNYHLKTSGTSYLQALKIIAQKAPDFFKEIWKTCLDKRTEMDKYYHLSCDPFSVPKDLKPIEYLNNPDARQTLHVSYMFVLNPKYDFRNRFFEILTKYENEYHIE VAEHIKRHVKELKIPEKIQN 369pA07097 MFTILPKKGISLGLGDRIGIATPGHIKVAKKYNFFPVFAQQ PetrotogaSIRELNFTGRTFTDVRKDVLNALVEENYVGNSGFDGDHL miothermaKSDEEIQYALDSGITMLTLDCSEHMNKDSSIKDQIFEQFYNKSFFVNDMPIEYSDKNELNKIVSIYASVIERVIDVWNKFPKVNKKEVTFEVSVDETDVPTDEKTHFLISKYIYDEGVKIDTLAPRFPGEFQKAIDYIGNIQEFKKSLIKQNKIAKYFGYRLSIHSGSDKFSIYPIIGEVTQGNYHLKTSGTSYLEAIKVVAQKDPEFFKKIWQTCLDKREEMDKYYHLSCDPFSVPKDLSPTEYLKNIDARQTLHVSYMFVLNPQYDFREKFFEILTKYQNEYY ENVANHIEKHVKELKIEETK 370pA07098 MFTILPKKGISIGLGDRIGIATPGHIKVAKKYNFFPVFAQQ Petrotoga oleariaSIRELNFTGRTFRDVRKDVLNALVEENYVGNSGFDGDHLKSDEEIQYALDSGITMLTLDCSEHMNKDSSVKESIFDQFYNKSFFVNDMPIEYSDKNELNKIVSIYGGVIERVIDVWNKFPKVNKKEVSFEVSVDETDVPTDEKTHFLISKYIYDEGVKIDTLAPRFPGEFQKGIDYIGNIQEFKKSLIKQDKIAKYFGYRLSIHSGSDKFSIYPIIGEVTQGNYHLKTSGTSYLEAIKVIAQKDPEFFKKIWQTCLDKREEMDKYYHLSCDPFSVPKDLSPTEYLQNPDARQTLHVSYMFVLNPQYDFRKKFFEILTKYQNEY HENVAHHIEKHVKELKVEETK 371pA07099 MKKLAKYSFGMGDRFAHQASWQLKAITEIEKQGIEVTPV ThermophagusWNKSNREHTTIGSKPEDNRDAACIKAIQKAGWSKPWYI xiamenensisDADHINLDTVDNFLESSDFFTIDVASYIGKKGDSKEEETFISKMKPLIGNLNIPGVNSPFKITEQQLRNIAGQYLHAAFMAGETYKYIESVKGKGNFITEVSMDEVPEPQTPVELFFILAMLAHYGVPAQTIAPKFTGRFNKGVDYVGDIETFRKEYEANLMVIDYAIQKFGFPPELKLSIHSGSDKFSIYPVIKELSQKHNKGFHLKTAGTTWLEEVIGLAMAGGEALLFVKNIYSRALDNIEKLCAPYADVIDINTDNLPKLSEVNNWTGEEFANALRHVPDHPMYNPDLRQLIHVAYKLAAENINQFNSFLEQHSEIV GKCVFENLYKRHAERLFVFSN 372pA07100 MKQLERFSMGIGDRFGHQGKAQLEALAEAKHLGCTIIPV TreponemaWNKSYREHSIIHTEPGQVRKEADWAVAALGWQDPYHV caldariumDADHISMKTVDLFLDSSDFFTLDVADYTGKAADEASIARFVAKHQHCIGKLQIPGIEQPITITETTLTSVARKYLLAIQEAGKLYRHIANKKGPENFITEVSIDETDQPQGPEDLLFILAMIADEGIPAQTIAPKFTGRFNKGVDYVGNLTQFEREFNQDILVIAYASKEFGLPKNLKLSVHSGSDKFSIYPIIKKAIYTHHAGLHLKTAGTTWLEELIGLASAGDEGLRIVQQIYRETYYRFDELCAPYATVIDIHKDKLPEPNSVQAWNGETYAAALRHIENHPQYNPHFRQLLHVGYKVAAELGETYLKALEAHEHHIALQ VKENLLDRHIRPIFIQ 373 pA07101MLDTPRYLGKLPHLSVGVRLPEVFLEGIMSGFKTGNSAG ThermofilumGVMLSYHRETAPEYVINAPPGDFELTRGHTGTSIRHYIEA uzonenseSVAKAKEKGVVVEVEADHVSVSVSSEAVKRISGGGTHRVLSEEEVRSALKYIEDEIREAVSTRNIYFYTIDTCDLIDYSSEKIAVDELRTVFKDLYPASLIERYKDINVVVNGTRIRFDEEKVMRLSLKLMRSIDVSERIYRIIKEMTPWPFGIEIAFDETPVTSDPHELFFVLNELRTRGIPVDFIAPNVGFQKREDFTGDLETLHSRVKTLHEVASFFGSLLSFHSGSGSSPYSMKGKGVHDIIRRAAGGLFKYKISGVYFELLMQLMSRSDIPSVRRLYEEIYDAVIELLEDQVKRKGELYDEVLVKRLEEHRKKSLNGYVRDSESPVFRYYSFLALNIRRNGERYLRNAIVELYLEDKGFREQV DREISALTVAFLDSLGFRGNVRLLR

Furthermore, the present disclosure provides polynucleotides encodingany of the polypeptides disclosed herein. The present disclosure alsoprovides constructs, vectors, plasmids that comprises thepolynucleotides. An expression vector carrying the polynucleotides ofthe disclosure may be any vector which is capable of expressingpolypeptides having D-fructose C4-epimerase activity in the selectedhost organism, and the choice of vector will depend on the host cellinto which it is to be introduced. Thus, the vector may be anautonomously replicating vector, i.e. a vector which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid, a bacteriophage or anextrachromosomal element, a minichromosome or an artificial chromosome.Alternatively, the vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome.

In the vector, the polynucleotides encoding for the D-fructoseC4-epimerase active polypeptide should be operably combined with asuitable promoter sequence. The promoter may be any DNA sequence whichconfers transcriptional activity to the host organism of choice and maybe derived from genes encoding proteins which are either homologous orheterologous to the host organism. Examples of suitable promoters fordirecting the transcription of the DNA fragment of the disclosure in abacterial host are the promoter of the lac operon of E. coli, thepromoter of the T7 RNA polymerase of T7 bacteriophage, the Streptomycescoelicolor agarase gene dagA promoters, the promoters of the Bacilluslicheniformis α-amylase gene (amyL), the promoters of the Bacillusstearothermophilus maltogenic amylase gene (amyM), the promoters of theBacillus amyloliquefaciens α-amylase gene (aqyQ), the promoters of theBacillus subtilis xylA and xylB genes.

The host organism of the disclosure either comprising a polynucleotideor an expression vector as described above is advantageously used as ahost cell in the recombinant production of a polypeptide according tothe disclosure. The cell may be transformed with a DNA constructcomprising the gene coding for the polypeptide of the disclosure or,conveniently by integrating the DNA construct into the host chromosome.Such an integration is generally considered to be advantageous as theDNA fragment is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may becarried out according to conventional methods such as e.g. by homologousor heterologous recombination or by means of a transposable element.Alternatively, the host organism may be transformed with an expressionvector as described above.

In accordance with the disclosure, the host organism may be a cell of ahigher organism such as an animal cell, including a mammal, an avian oran insect cell, or a plant cell. However, in preferred embodiments, thehost organism is a microbial cell, e.g. a bacterial or a fungal cellincluding a yeast cell.

In some embodiments, the foregoing polypeptide or microorganismexpressing the polypeptide is immobilized. In some embodiments, thepolypeptide or the microorganism expressing the polypeptide isimmobilized to a carrier or support. In some embodiments, thepolypeptide or the microorganism expressing the polypeptide isimmobilized to the carrier or support through adsorption, covalentattachment, non-covalent attachment, ionic interaction, entrapment,cross-linking, or metal-linking. In some embodiments, the carrier orsupport is an organic composition. In some embodiments, the organiccomposition is a natural polymer. In some embodiments the naturalpolymer is alginate, chitosan, chitin, collagen, carrageenan, gelatin,cellulose, starch, pectin, or sepharose. In some embodiments, theorganic composition is a synthetic polymer. In some embodiments, thesynthetic polymer is polystyrene, styrene divinylbenzene, polyvinylchloride, polyacrylate, polyamide, polypropylene, diethylaminoethylcellulose (DEAE cellulose), UV-activated polyethylene glycerol, ormethacrylate. In some embodiments, the organic composition isfunctionalized with a chemical group to facilitate enzymeimmobilization. In some embodiments, the functional group is an epoxygroup, an amino group, a hydrocarbon, a tertiary amine, a quaternaryamine, a carboxylic ester, nitrilotriacetic acid (NTA), iminodiaceticacid (IDA), aminophosphonic acid (AMPA), polyamine, or a carboxylicacid. In some embodiments, the carrier or support is an inorganiccomposition. In some embodiments, the inorganic composition is alumina,Zeolite, ceramics, celite, glass, silica, activated carbon, or charcoal.In some embodiments, the polypeptide or the microorganism expressing thepolypeptide is immobilized without a carrier or support. In someembodiments, the polypeptide or the microorganism expressing thepolypeptide is cross-linked to itself and/or an inert feeder proteinusing glutaraldehyde.

III. Modified Microorganisms

In another aspect, the disclosure provides a microorganism expressingany of the polypeptides disclosed herein. In some embodiments, thedisclosure provides a microorganism expressing a polypeptide comprisingan amino acid sequence that is at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-444. In some embodiments, themicroorganism is modified. In some embodiments, wherein the modifiedmicroorganism is genetically modified. In some embodiments, the modifiedmicroorganism is non-naturally occurring.

In some embodiments, the modified microorganism is derived fromEscherichia coli, Corynebacterum glutamicum, Aspergillus oryzae, Pichiapastoris, Bacillus subtilis, Caldithrix abyssi, Anaerolinea thermophila,Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacterthermohydrosulfuricus, Caldicellulosiruptor kronotskyensis, Dictyoglomusturgidum, Caldilinea aerophila, Rhodothermus marinus, Methanohalobiumevestigatum, Clostridium cavendishii, Kosmotoga olearia, Butyricicoccuspullicaecorum, Clostridium thermobutyricum, Litorilinea aerophila,Enterobacter mori., Caldisericum exile, Dictyoglomus thermophilum,Rhodothermus profundi, Caldibacillus debilis., Caloramator quimbayensis,Methanosalsum zhilinae, Pseudothermotoga thermarum, Pseudothermotogahypogea, Pseudothermotoga lettingae, Geosporobacter subterraneus,Melioribacter roseus, Lysinibacillus sphaericus, Clostridiumstercorarium, Truepera radiovictrix, Thermoflexus hugenholtzii,Petrotoga mobilis, Spirochaeta thermophila, Thermofilum pendens,Thermoanaerobacter siderophilus, Thermoanaerobacter mathranii,Thermoanaerobacter italicus, Thermoanaerobacterium xylanolyticum,Thermoanaerobacterium saccharolyticum, Ignisphaera aggregans, Thermotogamaritima, Caldanaerobacter subterraneus, Mesotoga infera, Thermosinuscarboxydivorans, Halanaerobium congolense, Halanaerobiumsaccharolyticum, Gracilibacillus halophilus, Caldicoprobacter faecalis,Thermoanaerobacter uzonensis, Lactobacillus ingluviei, Petrotogamexicana, Defluviitoga tunisiensis, Petrotoga miotherma, Petrotogaolearia, Thermophagus xiamenensis, Treponema caldarium, Thermofilumuzonense.

The modified microorganism according to the disclosure may, if it isintended for direct addition to a product where it is desired to haveD-fructose C4-epimerase activity, e.g. during a manufacturing process,be provided in the form of a microbial culture, preferably in aconcentrate form. Thus, such a culture may advantageously contain themicrobial cell according to the disclosure in a concentration which ispreferably in the range of 10⁵ to 10¹² per g of culture. The culture maybe a fresh culture, i.e. a non-frozen suspension of the cells in aliquid medium or it may in the form of a frozen or dried culture, e.g. afreeze-dried culture. The microbial cell may also for specific purposesbe immobilized on a solid substrate.

In some embodiments, the foregoing microorganism expressing thepolypeptide is immobilized. In some embodiments, the microorganismexpressing the polypeptide is immobilized to a carrier or support. Insome embodiments, the microorganism expressing the polypeptide isimmobilized to the carrier or support through adsorption, covalentattachment, non-covalent attachment, ionic interaction, entrapment,cross-linking, or metal-linking. In some embodiments, the carrier orsupport is an organic composition. In some embodiments, the organiccomposition is a natural polymer. In some embodiments the naturalpolymer is alginate, chitosan, chitin, collagen, carrageenan, gelatin,cellulose, starch, pectin, or sepharose. In some embodiments, theorganic composition is a synthetic polymer. In some embodiments, thesynthetic polymer is polystyrene, styrene divinylbenzene, polyvinylchloride, polyacrylate, polyamide, polypropylene, diethylaminoethylcellulose (DEAE cellulose), UV-activated polyethylene glycerol, ormethacrylate. In some embodiments, the organic composition isfunctionalized with a chemical group to facilitate enzymeimmobilization. In some embodiments, the functional group is an epoxygroup, an amino group, a hydrocarbon, a tertiary amine, a quaternaryamine, a carboxylic ester, nitrilotriacetic acid (NTA), iminodiaceticacid (IDA), aminophosphonic acid (AMPA), polyamine, or a carboxylicacid. In some embodiments, the carrier or support is an inorganiccomposition. In some embodiments, the inorganic composition is alumina,zeolite, ceramics, celite, glass, silica, activated carbon, or charcoal.In some embodiments, the microorganism expressing the polypeptide isimmobilized without a carrier or support. In some embodiments, themicroorganism expressing the polypeptide is cross-linked to itselfand/or an inert feeder protein using glutaraldehyde.

IV. Immobilization

The immobilization of the foregoing polypeptides and microorganisms canbe achieved using techniques known in the art. Immobilized enzymes havea number of distinct advantages over soluble enzymes such as, forexample, their use in continuous conversion systems. Exemplary ofpublications which review the art directed to enzyme immobilization arethe following: Goldstein, in Fermentation Advances, Academic Press, NewYork, N.Y. (1969), pp. 391-424; Goldstein et al., Z. Anal. Chem., 243,pp. 375-396 (1968); Kay, Process Biochem., 3 (8), pp. 36-39 (1968); Tosaet al., Kagaku To Seibutsu, 7 (3), pp. 147-155 (1967); Silman et al.,Ann. Rev. Biochem., 35 (2), pp. 873-908 (1966); Gryszkiewicz, FoliaBiologica, 19 (1), pp. 119-150 (1971); Zaborsky, “Immobilized Enzymes”,CRC Press, Cleveland, Ohio (1973); Datta et al., 3 Biotech. “Enzymeimmobilization: an overview on techniques and support materials,” 3(1):1-9 (2013); Gotovtsev et al., “Immobilization of microbial cells forbiotechnological production: Modern solutions and promisingtechnologies,” Appl. Biochem. Microbiol., 51: 792 (2015).

From the above noted publications, it is apparent that a number ofenzyme immobilization techniques have been described. These techniquesinclude covalently bonding an enzyme to a suitable insoluble carrier orsupport, encapsulation of an enzyme within a material which isimpermeable to the enzyme but permeable to the substrate and theproducts of the catalyzed reaction, adsorption of an enzyme on aninsoluble carrier and entrapment of an enzyme within a porous polymericmaterial wherein the pores are of such a size that will provide freeaccess of the substrate and the catalyzed reaction products but whichare sufficiently small to prevent the escape of the enzyme.

In some embodiments, the polypeptides disclosed herein or microorganismexpressing the polypeptide is immobilized. In some embodiments, thepolypeptide or the microorganism expressing the polypeptide isimmobilized to a carrier or support. In some embodiments, thepolypeptide or the microorganism expressing the polypeptide isimmobilized to the carrier or support through adsorption, covalentattachment, non-covalent attachment, ionic interaction, entrapment,cross-linking, or metal-linking.

In some embodiments, the carrier or support is an organic composition.In some embodiments, the organic composition is alginate, chitosan,chitin, collagen, carrageenan, gelatin, cellulose, starch, pectin,sepharose, polystyrene, styrene divinylbenzene, polyvinyl chloride,polyacrylate, polyamide, polypropylene, diethylaminoethyl cellulose(DEAE cellulose), UV-activated polyethylene glycerol, or methacrylate.In some embodiments, the organic composition is functionalized with achemical group. In some embodiments, the chemical group is an epoxygroup, an amino group, a hydrocarbon, a tertiary amine, a quaternaryamine, a carboxylic ester, nitrilotriacetic acid (NTA), iminodiaceticacid (IDA), aminophosphonic acid (AMPA), polyamine, or a carboxylicacid. In some embodiments, the organic composition is a natural polymer.In some embodiments the natural polymer is alginate, chitosan, chitin,collagen, carrageenan, gelatin, cellulose, starch, pectin, or sepharose.In some embodiments, the organic composition is a synthetic polymer. Insome embodiments, the synthetic polymer is polystyrene, styrenedivinylbenzene, polyvinyl chloride, polyacrylate, polyamide,polypropylene, diethylaminoethyl cellulose (DEAE cellulose),UV-activated polyethylene glycerol, or methacrylate. In someembodiments, the organic composition is functionalized with a chemicalgroup to facilitate enzyme immobilization. In some embodiments, thefunctional group is an epoxy group, an amino group, a hydrocarbon, atertiary amine, a quaternary amine, a carboxylic ester, nitrilotriaceticacid (NTA), iminodiacetic acid (IDA), aminophosphonic acid (AMPA),polyamine, or a carboxylic acid.

In some embodiments, the carrier or support is an inorganic composition.In some embodiments, the inorganic composition is alumina, zeolite,ceramics, celite, glass, silica, activated carbon, or charcoal. In someembodiments, the polypeptide or the microorganism expressing thepolypeptide is immobilized without a carrier or support. In someembodiments, the polypeptide or the microorganism expressing thepolypeptide is cross-linked to itself and/or an inert feeder proteinusing a cross-linking agent. In some embodiments, the cross-linkingagent is glutaraldehyde.

V. Methods for Producing Tagatose

The present invention provides methods and compositions for convertingfructose to tagatose. An economic biocatalytic process for conversion offructose to tagatose will likely require an enzyme that remains activefor extended periods of time at elevated temperatures. Elevatedtemperatures prevent contamination, can increase substrate solubilityand can increase reaction rate. Enzymes from thermophiles arenecessarily thermostable and active at elevated temperatures due totheir organism's native environment. Therefore, enzymes fromthermophiles were screened for D-fructose C4-epimerase (FC4E) activity.

In another aspect, the disclosure provides a method of producingtagatose, the method comprising: (a) contacting fructose with any of thepolypeptides disclosed herein or a microorganism expressing thepolypeptide; and (b) converting fructose to tagatose. In someembodiments, the method comprises: (a) contacting fructose with apolypeptide or a microorganism expressing the polypeptide, wherein thepolypeptide comprises an amino acid sequence that is at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1-444; and (b)converting fructose to tagatose. In some embodiments, step (b) convertsfructose to tagatose through C4-epimerization of fructose. In someembodiments, the polypeptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-444. In some embodiments, thepolypeptide consists essentially of an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 1-444. In some embodiments, thepolypeptide consists of an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1-444. In some embodiments, the polypeptide iscapable of converting fructose to tagatose through C4-epimerization offructose. In some embodiments, the polypeptide has D-fructoseC4-epimerase activity.

In one aspect, the disclosure provides a method for producing a tagatosecomposition, comprising the steps of: (a) providing a startingcomposition comprising greater than about 0.3%, about 0.5%, about 1%,about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90% offructose by weight; (b) contacting the starting composition with any ofthe polypeptide disclosed herein or a microorganism expressing thepolypeptide; and (c) producing a tagatose composition comprisingtagatose. In some embodiments, the polypeptide is capable of convertingfructose to tagatose through epimerization at the carbon-4 position offructose.

In some embodiments, the tagatose composition comprises tagatose in anamount greater than about 0.1%, about 0.2%, about 0.3%, about 0.5%,about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, or about 40% by weight. In some embodiments, thetagatose composition comprises tagatose in an amount between about0.3-80 wt %. In some embodiments, the tagatose composition comprisestagatose in an amount between about 0.5-60 wt %. In some embodiments,the tagatose composition comprises tagatose in an amount between about1-40 wt %.

In some embodiments, the starting composition comprises fructose in anamount between about 0.3% to about 70% by weight. In some embodiments,the starting composition comprises fructose in an amount between about10% to about 60% by weight. In some embodiments, the startingcomposition comprises fructose in an amount between about 20% to about60% by weight. In some embodiments, the starting composition comprisesfructose in an amount between about 20% to about 40% by weight. In someembodiments, the starting composition comprises fructose in an amountbetween about 30% by weight.

In some embodiments, contacting fructose with the polypeptide or themicroorganism expressing the polypeptide is performed at a temperaturebetween about 40° C. and about 100° C. In some embodiments, contactingfructose with the polypeptide or the microorganism expressing thepolypeptide is performed at a temperature between about 50° C. and about90° C. In some embodiments, contacting fructose with the polypeptide orthe microorganism expressing the polypeptide is performed at atemperature between about 60° C. and about 80° C. In some embodiments,the contacting of fructose with the polypeptide or the microorganismexpressing the polypeptide is performed at a temperature of about 60° C.

In some embodiments, the contacting of fructose with the polypeptide orthe microorganism expressing the polypeptide is performed at betweenabout pH 4.5 and about pH 8. In some embodiments, the contacting offructose with the polypeptide or the microorganism expressing thepolypeptide is performed at about pH 7.5.

In some embodiments, the contacting of fructose with the polypeptide orthe microorganism expressing the polypeptide is performed in thepresence of a metal ion. In some embodiments, the metal ion is adivalent metal cation. In some embodiments, the metal ion is Zn²⁺, Co²⁺,or Ni²⁺.

In some embodiments, the method disclosed herein is a batch process. Insome embodiments, the method disclosed herein is a continuous process.In some embodiments, the continuous process uses a fixed bed orfluidized bed reactor.

In some embodiments, the tagatose product is separated from the feedstock (fructose, glucose, and/or sucrose, etc.) through chromatographicmeans. In some embodiments, the tagatose is isolated from the feed usinga chromatographic method. In some embodiments, the chromatographicmethod is simulated moving bed chromatography.

In some embodiments, the isolated tagatose is further purified withcrystallization. In some embodiments, evaporative crystallization isused to make crystalline tagatose. In some embodiments, the tagatoseproduct is crystallized. In some embodiments, the evaporativecrystallization process is isothermal, and in another embodimentevaporative cooling is used. In some embodiments, the crystallization isisothermal evaporative crystallization or evaporative coolingcrystallization. In some embodiments, the fructose is produced fromglucose or sucrose.

In some embodiments, the polypeptide or the microorganism expressing thepolypeptide is immobilized to a carrier or support. In some embodiments,In some embodiments, the polypeptide or the microorganism expressing thepolypeptide is immobilized to the carrier or support through adsorption,covalent attachment, non-covalent attachment, ionic interaction,entrapment, cross-linking, or metal-linking. In some embodiments, thecarrier or support is an organic composition. In some embodiments, theorganic composition is alginate, chitosan, chitin, collagen,carrageenan, gelatin, cellulose, starch, pectin, sepharose, polystyrene,styrene divinylbenzene, polyvinyl chloride, polyacrylate, polyamide,polypropylene, diethylaminoethyl cellulose (DEAE cellulose),UV-activated polyethylene glycerol, or methacrylate. In someembodiments, the organic composition is functionalized with a chemicalgroup. In some embodiments, the chemical group is an epoxy group, anamino group, a hydrocarbon, a tertiary amine, a quaternary amine, acarboxylic ester, nitrilotriacetic acid (NTA), iminodiacetic acid (IDA),aminophosphonic acid (AMPA), polyamine, or a carboxylic acid.

In some embodiments, the carrier or support is an inorganic composition.In some embodiments, the inorganic composition is alumina, zeolite,ceramics, celite, glass, silica, activated carbon, or charcoal. In someembodiments, the polypeptide or the microorganism expressing thepolypeptide is cross-linked by a cross-linking agent. In someembodiments, the cross-linking agent is glutaraldehyde.

In some embodiments, the fructose is produced from glucose or sucrose.As a result, the present disclosure can provide a method for producingtagatose in high yield using common and inexpensive raw materials suchas glucose, fructose, sucrose, and the like, which enables massproduction of tagatose.

Accordingly, the present disclosure may further include hydrolyzingsucrose or isomerizing glucose to produce fructose prior to reaction offructose with the composition according to any one of embodiments of thepresent disclosure. Enzymes utilized in hydrolysis may beβ-D-fructosidase including β-fructofuranosidase, invertase, saccharase,sucrase, α-glucosidase, or α-D-glucohydrolase, without being limitedthereto. Examples of the enzyme isomerizing glucose may include glucose(xylose) isomerase and phosphoglucoisomerase, without being limitedthereto.

EXAMPLES

The following working examples are illustrative and are not intended tobe limiting, and it will be readily understood by one of skill in theart that other embodiments may be utilized.

Example 1 Preparation of Recombinant Microorganisms Producing D-Fructose4-Epimerase (FC4E)

The genomes of thermophilic organisms were searched for enzymes withputative FC4E activity. Two protein scaffolds were identified, andpolynucleotides encoding amino acid sequences SEQ ID NO:1 to SEQ IDNO:23 (Scaffold1) and SEQ ID NO:321 to SEQ ID NO:373 (Scaffold2) derivedfrom 56 thermophilic microorganisms (Table 3) were synthesized(GenScript) and inserted into the pARZ4 expression vector, generatingthe corresponding recombinant vectors: pA06233, pA06234, pA06235,pA06236, pA06237, pA06238, pA06239, pA06240, pA06241, pA07068, pA07069,pA07070, pA07071, pA07072, pA07073, pA07074, pA07075, pA07076, pA07077,pA07078, pA07079, pA07080, pA07081, pA06242, pA06243, pA06246, pA06247,pA06248, pA06249, pA06250, pA06252, pA06253, pA06254, pA06255, pA06256,pA06257, pA06261, pA06265, pA06266, pA06267, pA06268, pA06270, pA06271,pA06272, pA06273, pA06274, pA07082, pA07083, pA07084, pA07085, pA07086,pA07087, pA07088, pA07089, pA07090, pA07091, pA07092, pA07094, pA07095,pA07096, pA07097, pA07098, pA07099, pA07100, pA07101. The recombinantvectors were used in a heat shock method to transform competent E. coli(NEBT7EL; New England Biolabs), thereby preparing recombinantmicroorganisms.

Each transformed recombinant microorganism was inoculated into 1 mlLB-kanamycin medium, cultured by shaking at 37° C. overnight. Theculture was inoculated to 5 ml TB-kanamycin medium and grown for 2 hoursat 37° C., followed by 25° C. for 1 hour. The culture was induced with50 uL 50 mM IPTG and grown overnight. Finally, the culture wascentrifuged at top-speed for 5-minutes and stored at −80° C.

The prepared E. coli recombinant microorganisms were named asNEBT7EL-pA06233, NEBT7EL-pA06234, NEBT7EL-pA06235, NEBT7EL-pA06236,NEBT7EL-pA06237, NEBT7EL-pA06238, NEBT7EL-pA06239, NEBT7EL-pA06240,NEBT7EL-pA06241, NEBT7EL-pA07068, NEBT7EL-pA07069, NEBT7EL-pA07070,NEBT7EL-pA07071, NEBT7EL-pA07072, NEBT7EL-pA07073, NEBT7EL-pA07074,NEBT7EL-pA07075, NEBT7EL-pA07076, NEBT7EL-pA07077, NEBT7EL-pA07078,NEBT7EL-pA07079, NEBT7EL-pA07080, NEBT7EL-pA07081, NEBT7EL-pA06242,NEBT7EL-pA06243, NEBT7EL-pA06246, NEBT7EL-pA06247, NEBT7EL-pA06248,NEBT7EL-pA06249, NEBT7EL-pA06250, NEBT7EL-pA06252, NEBT7EL-pA06253,NEBT7EL-pA06254, NEBT7EL-pA06255, NEBT7EL-pA06256, NEBT7EL-pA06257,NEBT7EL-pA06261, NEBT7EL-pA06265, NEBT7EL-pA06266, NEBT7EL-pA06267,NEBT7EL-pA06268, NEBT7EL-pA06270, NEBT7EL-pA06271, NEBT7EL-pA06272,NEBT7EL-pA06273, NEBT7EL-pA06274, NEBT7EL-pA07082, NEBT7EL-pA07083,NEBT7EL-pA07084, NEBT7EL-pA07085, NEBT7EL-pA07086, NEBT7EL-pA07087,NEBT7EL-pA07088, NEBT7EL-pA07089, NEBT7EL-pA07090, NEBT7EL-pA07091,NEBT7EL-pA07092, NEBT7EL-pA07094, NEBT7EL-pA07095, NEBT7EL-pA07096,NEBT7EL-pA07097, NEBT7EL-pA07098, NEBT7EL-pA07099, NEBT7EL-pA07100, andNEBT7EL-pA07101.

Example 2 Purification and Measurement of D-Fructose 4-EpimeraseActivity

2-1. Purification of D-Fructose 4-Epimerase

The 76 microorganisms expressing D-fructose C4-epimerases created inExample 1 were dissolved in lysis buffer (lysozyme, DNAseI, Bugbuster,300 mL 20 mM PO₄ pH 7.5, 500 mM NaCl, and 20 mM Imidazole). Two to threeglass beads were added to each well and were disrupted by shaking at 25°C. and 220 rpm for 30 minutes. The disrupted liquid was centrifuged at2200×g for 6-10 minutes. The obtained supernatant was loaded onto aNi-NTA plate and shaken for 10 minutes at room temperature. The platewas centrifuged for 4 minutes at 100×g followed by two washes of 500 uLbinding buffer (300 mL 20 mM PO₄ pH 7.5, 500 mM NaCl, 20 mM Imidazole)and two minute centrifugation (500×g). The proteins were eluted with 150uL elution buffer (15 mL 20 mM PO₄ pH 7.5.5, 500 mM NaCl, 500 mMImidazole) and shaken for 1 minute at 0.25 maximum shaking speedfollowed by centrifugation for 2 minutes at 500×g. The recovered proteinwas desalted into a buffer solution for enzyme activity evaluation(Scaffold1: 20 mM KPO₄, 50 mM NaCl, 300 uM CoCl₂, pH 7.5; Scaffold2: 20mM KPO₄, 50 mM NaCl, 300 uM NiCl₂, pH 7.5).

2-2. Measurement of D-Fructose 4-Epimerase Activity

In order to identify the activity of the 76 D-fructose C4-epimerasespurified in Example 2-1, the enzymes were reacted with fructosesubstrate at 60° C. The reaction was started by reacting 20 mM fructosewith purified enzyme in buffer (Scaffold1: 20 mM KPO₄, 50 mM NaCl, 300uM CoCl₂, pH 7.5; Scaffold2: 20 mM KPO₄, 50 mM NaCl, 300 uM NiCl₂, pH7.5) and allowed to react overnight, followed by quenching the reactionproduct by placing the reaction at −80° C.

All monosaccharides were analyzed using an Agilent 6545 q-tof run innegative/high sensitivity mode. Sugars were separated using a VarianHi-plex Pb++ column (100 mm×7.7 mm). A 4:1 mixture of methanol andchloroform was added to the reference mix to induce the formation of Cl+adducts. Quantification was performed on the (M+Cl)− adduct of 215.0320m/z. Standards of fructose and tagatose were purchased from SigmaAldrich and calibration was performed from 1 to 1000 ppM.

The D-fructose C4-epimerases successfully produced tagatose fromfructose. The relative conversion rates are shown in FIG. 1 (Scaffold1)and FIG. 2 (Scaffold2). Specific Q-tof traces for a subset of the FC4Esare shown in FIG. 3 and FIG. 4.

2-3. Activity Analysis of Alternate Metal Ions.

In order to determine the dependence of epimerase activity on metal salttype, a subset of the Scaffold1 purified enzymes from Example 2-1 weretested for activity with alternative metal salts: 0.3 mM NiCl₂ and 0.3mM ZnCl₂ (FIG. 5). All conditions were the same as in Example 2-2 exceptCoCl₂ was substituted with an alternate metal salt.

Example 3 Activity of D-Fructose 4-Epimerases in High Sugar

In Example 2-2, the putative D-fructose C4-epimerases were screened at alow sugar concentration to determine if any activity was present.However, an industrially useful enzyme should be active at highersubstrate concentrations. Therefore, the FC4Es were challenged with 1 Mfructose. The experiments were conducted similarly to those in Example2-2, except 1 M fructose was used as substrate and the reaction waslimited to 20 minutes instead of overnight. Several FC4Es showedactivity in these more challenging conditions (FIG. 6).

Example 4 Heat Stability of D-Fructose 4-Epimerases

Eight FC4Es pA06233-pA06240 were tested for heat stability. Enzymes wereexpressed and purified similar to Example 2-1. The FC4Es were incubatedat 60° C. for 24 hours and then each enzyme was reacted with fructose(final concentration of 0.57M) for 20 minutes at 60° C. The conversionof fructose to tagatose was measured as described in Example 2-2. Therelative tagatose yield for the enzymes can be seen in FIG. 7.

Example 5 Characterization of Top D-Fructose 4-Epimerases

Top FC4Es were further characterized to determine how their activityvaried with temperature, sugar concentration, reaction time, and proteinconcentration. To determine the temperature optimum, enzymes pA06234,pA06235, pA06238 were purified as in Example 2-1 and reacted with 1.9 Msugar for 20 minutes in buffer (20 mM KPO₄, 50 mM NaCl, 300 uM CoCl₂, pH7.5) at the following temperatures: 50° C., 60° C., 65° C., 70° C., 80°C., 90° C. and 100° C. The conversion of fructose to tagatose wasmeasured the same as Example 2-2. The relative yield of tagatose overthe measured temperature range is shown in FIG. 8.

Next, the conversion of fructose over time was measured for FC4EspA06234, pA06235, pA06238 and pA07079. Purified protein was obtained asin Example 2-1. FC4E was reacted with 1.9 M fructose in 20 mM KPO₄, 50mM NaCl, 300 uM CoCl₂, pH 7.5 for 0-24 hours at 60° C. The conversion offructose to tagatose was measured as in Example 2-2. The relative yieldfor the measured reaction times is shown in FIG. 9.

The effect of protein concentration was determined for FC4Es pA06234,pA06235, pA06238 and pA07079. Purified protein was obtained as inExample 2-1. The protein was then concentrated using Vivaspin 6, 5 kDamolecular weight protein concentrators. Five different dilutions of eachFC4E was reacted with 1.9 M fructose in 20 mM KPO₄, 50 mM NaCl, 300 uMCoCl₂, pH 7.5 at 60° C. for 20 minutes. The conversion of fructose totagatose was measured as in Example 2-2. The relative yield for thedifferent protein concentrations is shown in FIG. 10.

The effect of substrate concentration on fructose to tagatose conversionwas measured for pA06238. Purified protein was obtained as in Example2-1. pA06238 was reacted with 50 mM-2.8 M fructose in 20 mM KPO₄, 50 mMNaCl, 300 uM CoCl₂, pH 7.5 for 20 minutes at 60° C. The conversion offructose to tagatose was measured as in Example 2-2. The relativetagatose yield for the tested sugar concentrations is shown in FIG. 11.

Example 6 Thermostability of pA06238

An industrially useful enzyme will likely need to operate at sustainedelevated temperatures. Example 4 demonstrates that pA06238 is stablewhen incubated at 60° C. for a day. To further investigate thethermostability of pA06238, the lifetime of pA06238 at 80° C. wasinvestigated. Purified protein was obtained as in Example 2-1. pA06238was incubated at 80° C. for 0-72 hours. Following incubation, the enzymewas reacted with 1.9 M fructose in 20 mM KPO₄, 50 mM NaCl, 300 uM CoCl₂,pH 7.5 for 20 minutes at 60° C. The conversion of fructose to tagatosewas measured as in Example 2-2. The relative tagatose yield for thetested sugar concentrations is shown in FIG. 12. After 3 daysincubation, there was not a statistically significant loss of activitycompared to the early timepoints.

Example 7 Improved Expression of pA06238 Through Coding SequenceOptimization

We used our in-house DNA optimization software, Scribe™, to improveexpression of pA06238. Thirty optimized polynucleotide sequences thatcode for the pA06238 amino acid sequence inserted in the pARZ4expression vector were ordered from Twist Bioscience. The plasmids weretransformed into NEBT7EL and the FC4Es were expressed and purified as inExample 2-1. Purified enzyme was reacted in buffer (20 mM KPO₄, 50 mMNaCl, 300 uM CoCl₂, pH 7.5) with 1.9 M fructose at 60° C. for 24 hours.To detect the formation of tagatose, reaction assays were diluted20-fold with water. Plates were sealed and placed into a Waters AQUITY-HUPLC system consisting of an FTN samples manager, RI and UV detector.Separation of tagatose from fructose was conducted on an Agilent Hi-plexPb 150×4.0 mm column (p/n PL1115-9999) with 100% water at 85° C. at aflow rate of 0.45 ml/min. Quantification was done using the RI detectorchannel. The purified protein yield and corresponding enzyme activityfor the 30 optimized polynucleotide sequences compared to the parentpA06238 (square) is shown in FIG. 13. The most improved variant improvedexpression by 1.32-fold with a corresponding 1.35 fold improvement intagatose yield.

Example 8 Immobilization of pA06238 on Commercially Available Supports

8-1. Enzyme Immobilization Resins

The FC4E pA06238 enables a one-step enzymatic process to convertfructose to tagatose. An example biocatalytic process for makingtagatose from fructose involves the use of immobilized FC4E in a fixedbed reactor or fluidized bed reactor. We successfully demonstrated theimmobilization of pA06238 on several commercially available supports.

Enzyme immobilization resins were purchased from Chiralvision (IB-ADS-1,IB-ADS-2, IB-ADS-3, IB-ADS-4, IB-ANI-1, IB-ANI-2, IB-ANI-3, IB-ANI-4,IB-CAT-1, IB-COV-1, IB-COV-2, IB-COV-3) and Purolite (ECR1030M,ECR1090F, ECR1504, ECR1640, ECR8204F, ECR8209F, ECR8285, ECR8309F,ECR8409F, ECR8806F). These resins vary in composition (methacrylate,styrene or polyacrylic) as well as functional group (epoxy, amino,octadecyl, tertiary amine, quaternary amine, carboxylic ester, phenyl,sulphonic, or no functional group).

The standard protocol for each resin from each company was used toimmobilize pA06238 onto 100 mg resin. Two different volumes of enzyme(400 uL and 200 uL) were incubated with resin at room temperature toimmobilize the enzyme onto the support. After incubation, theimmobilized enzyme was washed five times with either water(Chiralvision) or buffer (Purolite). The immobilized enzyme was reactedwith 1 M fructose in buffer at 60° C. for 20 minutes. The relativeactivity obtained for each support is shown in FIG. 14.

8-2. Metal Chelating Resins

Since pA06238 contains a hexahistidine tag, the enzyme should immobilizeonto loaded metal chelating resins through a metal-hexahistidine taginteraction. The following commercial metal chelating resins wereobtained to test enzyme immobilization: Ni-NTA agarose (Qiagen), NuviaIMAC (Bio-Rad), S930 plus (Purolite), S940 (Purolite), S950 (Purolite),Lewatit MDS TP 208 (Lanxess), Lewatit MDS TP 260 (Lanxess), Lewatit MDSTP 260 (Lanxess), Relite MAC5 (Mitsubishi), Relite MAC1 (Mitsubishi),DIAION CR11 (Mitsubishi), DIAION CR20 (Mitsubishi), Amberlite IRC748(Dow), and Amberlite IRC747 (Dow). The metal chelating resins utilizeone of the following functional groups for metal binding:nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), aminophosphonicacid (AMPA), polyamine, or carboxylic acid. The NTA resins werepre-loaded with nickel. For the remaining resins, copper was loaded ontothe resin by incubating 100 mg of resin with 400 uL of 200 mM CuSO₄followed by washing with 100 mM sodium acetate. To immobilize pA06238,all resins were washed with buffer (20 mM KPO₄, 50 mM NaCl, 300 uMCoCl₂, pH 7.5) and 8 mg of protein was incubated with 100 mg for 18hours with mixing and let stand for 20 hours at room temperature.Immobilized enzyme was washed with buffer to remove unbound protein, andthen assayed for fructose to tagatose conversion by reacting with 1 Mfructose for 20 minutes at 60° C. Four metal chelating resins haddetectable activity, but all were lower than the best enzymeimmobilization resins from Example 8-1 (Table 4).

TABLE 4 Relative activity of pA06238 immobilized on metal chelatingresins compared to ECR8204F Resin Percent of ECR8204F Yield QiagenNi-NTA 18.1% Nuvia IMAC 16.0% Amberlite IRC747  6.7% DIAION CR20  5.6%

Example 9 Conversion of Fructose to Tagatose in a Fixed Bed Reactor

NEBT7EL-pA06238 was grown on LB with 50 μg/ml kanamycin. A 600 mlculture of TBkan5 was inoculated with NEBT7EL-pA06238 and incubatedovernight at 37° C. at 200 rpm. The next morning, a 10 L fermentor wasprepared with 9.5 L of TB and then inoculated with 500 ml of theovernight culture. The culture was grown at 37° C. The pH was maintainedat 6.2 with NaOH and the dO₂ was maintained ≥20%. After 2 hours ofgrowth, the temperature was dropped to 25° C. The culture was grown foran additional 1 hour with the OD₆₀₀ around 7. IPTG was added to a finalconcentration of 1 mM and CoCl₂ was added to 25 μM. Additional CoCl₂ wasadded 1 and 2 hours after induction to bring the final concentration to300 μM. The cells were grown for 20 hours at which point the fermentorwas chilled to 10° C. and the cells were harvested by centrifugation.The cell pellet was stored at −80° C. until use.

The cell pellet from the fermentation was lysed by stirring in bufferwith lysozyme and DNAse. Cell debris was removed by centrifugation andthe supernatant was filtered through a 0.45 micron filter. Filteredsupernatant was incubated with Ni-NTA agarose resin and then enzyme waseluted with imidazole. Purified FC4E pA06238 was immobilized onto 5.25grams of ECR8204F resin using the standard published protocol fromPurolite.

The immobilized enzyme was loaded into a 11×300 mm glass fixed bedreactor and run for approximately 200 h at constant temperature (60° C.)with a constant feed composition of 30 wt % fructose+70 wt % aqueousbuffer solution (20 mM KPO₄, 50 mM NaCl, 300 uM CoCl₂). Feed rate washeld constant at 140 uL/min throughout the run. The fixed bed reactionreached a maximal conversion of approximately 30% tagatose and had ahalf-life of −50 hours (FIG. 15).

Example 10 Simulated Moving Bed Separation of Tagatose from a MixedSugar Solution

Eight 25 mm×300 mm glass, jacketed chromatography columns were loadedwith Dowex™ monosphere 99 Ca/320 resin and connected to a temperaturecontrol system including a recirculating hot water system (Polyscience,AP20S150-A11B) and electrical heat-loss compensation capable oftemperature control to within ±1° C. The columns were plumbed in a3-zone simulated moving bed configuration using a Semba BiosciencesOctave chromatography system (SMB unit). A degassed feed sugar solutionof 10 wt % tagatose, 20 wt % fructose and 70 wt % purified water wasmetered into the SMB unit via a dual-piston positive displacement pump(Octave 100) including pre- and post-filters and pulse-dampening toensure smooth continuous flow at specified volumetric flow rates. Theeluent feed to the system was a degassed purified water solution,similarly, fed to the SMB unit using a larger dual-piston positivedisplacement pump (Octave 300) including pre- and post-filters andpulse-dampening. At steady-state, a smaller extract stream wascontinuously withdrawn from the unit using a third dual-piston positivedisplacement pump (Octave 100), while the larger raffinate stream flowedfrom the unit via a back-pressure control valve. Product rates weremonitored intermittently by manual volumetric measurement.

A range of experiments were performed to demonstrate purification oftagatose in the extract stream. In Condition 1 the SMB separation wasperformed on 1 L of the degassed sugar feed solution, collecting 3.6 Lof extract and 12.2 L of raffinate over 24 h. Table 5 shows that ahighly purified tagatose stream can be collected using SMB separation.

TABLE 5 Measured concentrations of tagatose and fructose during SMBCondition 1. Tagatose Fructose concentration, g/L Concentration, g/LExtract Stream 33.35 0.25 Raffinate Stream 1.87 31.30

Example 11 Isothermal Batch Crystallization of Tagatose from a MixedSugar Solution

A mixed sugar solution comprising 200 g tagatose, and 50 g fructose wassolvated in 120 g water by gently heating the solution with rotaryagitation using a rotary evaporator (Eyela, N-1200BS) to a temperatureof 60° C. Isothermal batch crystallization was performed by pullingvacuum of the evaporation flask to a constant pressure of 100 mmHg, thenslowly evaporating water from the flask under constant vacuum over a 270min period. Once 20 ml of condensate had been collected, the experimentwas paused briefly, 0.25 g of pure tagatose seed crystals were added tothe flask, and then vacuum conditions were restored, and the evaporationwas continued. Slow crystallization was observed. The experiment wascontinued until 80 ml condensate had been collected, after which theflask was quickly disconnected from the rotary evaporator and the slurryof mother liquor and crystals was quickly filtered via a Buchner funnelfitted with a cellulose filter. The collected crystals were washed with62.5 g of ice-cold water, then dried under vacuum at 60° C. for 2 h. Thefinal tagatose crystals contained 98.51 wt % tagatose and 1.49 wt %fructose, a significant improvement versus the feed mixture.

Example 12 Active Site Mutants Improve FC4E Activity

12-1. Active Site Mutants of pA06234

Active site mutants of pA06234 were generated to determine the impact ofthese residues on catalysis. For each mutant, primer pairs were designedto 1) open the parent CDS at the desired insertion point (omitting thecodon intended to be replaced) and 2) split the plasmid backbone withinthe antibiotic selection marker. Polymerase chain reaction (PCR) thensuccessfully generated two linear fragments from each plasmid. The new,desired codon was embedded in the forward primer of the first PCR.Primers were designed to also include flanking homology. Subsequenttwo-way flanking homology-based DNA assembly produced plasmids used totransform our standard cloning strain. After confirming DNA sequence bySanger sequencing, sequence-perfect plasmids were used to transformNEBT7EL.

FC4E active site mutants (SEQ ID NOs: 24-45) were expressed and purifiedas in Example 2-1. Purified enzyme was reacted with 1.9 M fructose inbuffer (20 mM KPO₄, 50 mM NaCl, 300 uM CoCl₂) overnight at 60° C.Conversion of fructose to tagatose was measured as in Example 2-2 (FIG.16). Promising active site mutants with tagatose conversion similar toor better than the parent were further investigated by reacting withfructose for 20 minutes. Four mutants with increased activity comparedto the parent enzyme (pA06234) are shown in Table 6.

TABLE 6 Plasmid ID Mutation Fold Increase in Tagatose Yield pA07219Y361A 2.14 pA07210 E178A 2.14 pA07203 Y64F 1.40 pA07202 Y64A 1.18

12-2. Transferring pA06234 Active Site Mutations into pA06238

The native FC4Es (SEQ ID NOs: 1-23, 321-373) are sufficiently similar(FIG. 17) that beneficial mutations in one enzyme can be transferred tothe corresponding position in another FC4E enzyme and havecorrespondingly beneficial impacts. We demonstrate this by transferringthe top three mutations from Example 12-1 (Y64F, E178A, Y361A) into thepA06238 sequence (Y53F, D167A, Y340A; SEQ ID NO: 231). Thepolynucleotide sequence for SEQ ID NO: 231 was ordered from TwistBioscience. The plasmid was transformed, and the protein was expressedand purified as in Example 2-1. The purified enzyme was reacted with 1.9M fructose in buffer (20 mM KPO₄, 50 mM NaCl, 300 uM CoCl₂) for 24 hoursat 60° C. The mutant enzyme, SEQ ID NO: 231, had a 1.4-fold improvementin tagatose conversion compared to the parent enzyme, pA06238.

12-3. Active Site Mutants of pA06238

Forty-two active site residue positions in pA06238 were chosen forsite-saturation mutagenesis to create a library of pA06238 mutants.Mutant plasmids were generated similar to Example 12-1, except thatprimers were used that encoded all amino acids at the specified mutantresidue position, and clones were selected for use without sequenceverification.

FC4Es from the mutant library were expressed and purified similar toExample 2-1. Purified enzyme was reacted with 1.9 M fructose in buffer(20 mM KPO₄, 50 mM NaCl, 300 uM CoCl₂, pH 7.5) at 60° C. for 1 and 24hours. Conversion of fructose to tagatose was measured as in Example 7.Fold increase in percent tagatose conversion for each tested mutant isshown in FIG. 18 (1 hour data) and FIG. 19 (24 hour measurements). Thefollowing wild-type residue positions had mutant library members thatshowed increased conversion of tagatose compared to their parent FC4E:T43, P44, H45, Q46, Y53, D84, L89, Q92, H119, C122, S123, M124, T166,D167, V168, P169, I170, A171, G173, G174, E175, E176, G178, V210, M212,S248, T249, K269, V270, P272, L335, R337, R339, Y340. The top mutantsfrom the library were sequenced and are included here as SEQ ID NOs:371-444.

Example 13 Computational Design of pA06238 to Improve Expression andStability

Computational design of pA06238 guided by native sequence alignment wasused to improve protein expression and stability. In order for a givenamino acid at a specific residue position to be allowed in the finaldesign, it had to pass two filters: one based on native sequencealignment and one based on computational modeling. A position-specificscoring matrix (PSSM) was created from a native sequence alignment. Twodifferent alignments were used: 1) aligned sequences were only fromthermophilic organisms and 2) aligned sequences were allowed from anyorganism. Any amino acid with a positive score in the PSSM passed thefirst filter. For each passing amino acid, the Archytas™ protein designsoftware was used to predict if mutating to the given amino acid waspredicted to have a ΔΔG below a given cutoff. Several cutoffs, −2.5,−2.0, −1.5, −1.0, −0.5, were used to select the final designs. All aminoacids that passed both filters were combined in a final computationaldesign. Homology models of pA06238 built with Archytas™ were used asinput for both the ΔΔG calculations and final computational designs.

Ninety two designed enzymes were chosen for characterization (FIG. 20).The number of mutations per design ranged from 2-47 with an average of15 mutations per design. Mutations occurred at 138 residue positionswithin pA06238. A sequence alignment of the designs with all mutationshighlighted is shown in FIG. 20. Polynucleotide sequences for the 92designed enzymes (SEQ ID NOs: 46-136) were ordered from TwistBioscience. The plasmids were transformed, and the protein was expressedand purified as in Example 2-1. The purified FC4Es were reacted with 1.9M fructose in buffer (20 mM KPO₄, 50 mM NaCl, 300 uM COCl₂) for 24 hoursat 60° C. The fold change in total conversion (total purified proteinmultiplied by percent conversion to tagatose for a standard amount ofenzyme) is shown in FIG. 21.

Example 14 Computational Design of pA06238 for Increased Stability

Enzyme inactivation has been observed when enzymes are exposed toaldehydes (Jennewein et al., 2006; Lorenzo et al., 2007). Lysines and toa lesser extent cysteines are particularly susceptible to modificationby aldehydes, causing enzyme inactivation. The Archytas™ design softwarewas used to design mutants of pA06238 with a reduced number of exposedlysine and cysteine residues. 184 designed lysine mutant enzymes werechosen to be experimentally characterized. Half of the designed mutantsonly mutated lysine and cysteine residues (SEQ ID NOs: 137-231; FIG.22), and the other half allowed mutations surrounding the lysine andcysteine positions (SEQ ID NOs: 232-320; FIG. 23). The mutants wereordered from Twist Bioscience and tested for FC4E activity. The plasmidswere transformed, and the enzymes were expressed and purified as inExample 2-1. The purified FC4Es were reacted with 1.9M fructose inbuffer (20 mM KPO₄, 50 mM NaCl, 300 uM CoCl₂) for 24 hours at 60° C. Thefold change in tagatose conversion for a subset of lysine mutants isshown in FIG. 24.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andthe range of equivalency of the claims are intended to be embracedtherein.

1. A polypeptide comprising an amino acid sequence having at least 60%sequence identity to any one of SEQ ID NOS:1-444, wherein thepolypeptide has D-fructose C4-epimerase activity.
 2. The polypeptide ofclaim 1, wherein the polypeptide comprises one or more modifications atthe amino acid residue corresponding to position L3, S4, K5, D6, Y7, L8,R9, K10, K11, V13, Y14, S15, I16, C17, S18, S19, S21, Y22, E25, S27,E29, F30, K32, E33, K34, G35, D36, Y37, E41, T43, P44, H45, Q46, Q49,F50, Y53, S54, M56, E59, K62, N63, F64, M66, K67, K70, E71, K72, E75,E76, D77, K78, I80, D84, H85, L89, Q92, D93, E94, S96, P97, T98, N101,K102, K104, D105, R108, F110, S113, Y115, K116, K117, H119, D121, C122,S123, M124, P125, L126, S127, D128, D129, P130, K131, V132, P134, Y135,E136, K137, E140, R141, T142, R143, E144, F146, E147, I148, E150, E151,T152, A153, R154, K155, Y156, N157, F158, Q159, P160, V161, T166, D167,V168, P169, I170, A171, G172, G173, G174, E175, E176, E177, G178, V179,V182, D184, S187, A188, I189, S190, S191, L192, K193, K194, Y195, N197,D198, V199, P200, N201, I202, W203, D204, R205, G208, V210, I211, M212,L213, I215, G216, F217, S218, Y219, D220, K221, V222, E224, D228, K229,V230, R231, G232, I233, L234, E235, V237, K238, R239, E240, D241, L242,V244, E245, G246, H247, S248, T249, D250, A253, Y255, A256, R258, N259,E262, R266, K269, V270, G271, P272, A273, R280, G281, V282, L284, S286,N287, D290, E291, I292, E295, R296, E297, S299, N300, K302, R303, E307,D313, Y315, K318, Y319, K321, D322, S323, K324, R325, L326, E327, L328,I330, W331, N333, L334, L335, D336, R337, R339, Y340, E343, Y344, E345,K348, M349, V350, N352, K353, E356, N357, F358, E360, G361, V362, D363,I364, K365, F366, Y368, Q369, Y370, Y372, D373, S374, Y375, F376, D377,R379, E380, K382, M383, K384, N385, D386, R388, E389, K392, K393, K396,R397, E400, S403, N407, or L408 of SEQ ID NO:6; or combinations thereof.3. The polypeptide of claim 1, wherein the polypeptide comprises one ormore of modifications: (1) the amino acid residue corresponding toposition L3 of SEQ ID NO:6 is GLU, or THR; (2) the amino acid residuecorresponding to position S4 of SEQ ID NO:6 is ARG, ASN, ASP, GLN, GLU,LEU, MET, or PRO; (3) the amino acid residue corresponding to positionK5 of SEQ ID NO:6 is ALA, ARG, GLN, HIS, LEU, MET, PHE, TRP, or VAL; (4)the amino acid residue corresponding to position D6 of SEQ ID NO:6 isALA, ARG, ASN, GLN, GLU, HIS, or LEU; (5) the amino acid residuecorresponding to position Y7 of SEQ ID NO:6 is ASN, HIS, or TRP; (6) theamino acid residue corresponding to position L8 of SEQ ID NO:6 is ASN,or GLY; (7) the amino acid residue corresponding to position R9 of SEQID NO:6 is GLN, GLU, HIS, ILE, LEU, or THR; (8) the amino acid residuecorresponding to position K10 of SEQ ID NO:6 is ALA, ARG, ASN, ASP, GLU,LEU, MET, PRO, or VAL; (9) the amino acid residue corresponding toposition K11 of SEQ ID NO:6 is ARG, GLN, GLU, HIS, ILE, LEU, PHE, TRP,TYR, or VAL; (10) the amino acid residue corresponding to position V13of SEQ ID NO:6 is ILE; (11) the amino acid residue corresponding toposition Y14 of SEQ ID NO:6 is PHE; (12) the amino acid residuecorresponding to position S15 of SEQ ID NO:6 is ALA; (13) the amino acidresidue corresponding to position I16 of SEQ ID NO:6 is VAL; (14) theamino acid residue corresponding to position C17 of SEQ ID NO:6 is ALA,ARG, GLY, ILE, LEU, SER, THR, or VAL; (15) the amino acid residuecorresponding to position S18 of SEQ ID NO:6 is ALA, or ASN; (16) theamino acid residue corresponding to position S19 of SEQ ID NO:6 is ASN;(17) the amino acid residue corresponding to position S21 of SEQ ID NO:6is ARG, GLN, GLU, LYS, or PRO; (18) the amino acid residue correspondingto position Y22 of SEQ ID NO:6 is TRP; (19) the amino acid residuecorresponding to position E25 of SEQ ID NO:6 is ARG, or ILE; (20) theamino acid residue corresponding to position S27 of SEQ ID NO:6 is ALA,or CYS; (21) the amino acid residue corresponding to position E29 of SEQID NO:6 is ARG, GLN, ILE, or LEU; (22) the amino acid residuecorresponding to position F30 of SEQ ID NO:6 is ALA, HIS, or THR; (23)the amino acid residue corresponding to position K32 of SEQ ID NO:6 isALA, ARG, GLN, ILE, LEU, MET, or SER; (24) the amino acid residuecorresponding to position E33 of SEQ ID NO:6 is ARG, ASN, ASP, GLN, HIS,or THR; (25) the amino acid residue corresponding to position K34 of SEQID NO:6 is ARG, ASP, GLN, GLU, HIS, ILE, LEU, MET, PHE, SER, THR, TYR,or VAL; (26) the amino acid residue corresponding to position G35 of SEQID NO:6 is ASN, ASP, HIS, or SER; (27) the amino acid residuecorresponding to position D36 of SEQ ID NO:6 is ARG, ASN, GLN, GLU, HIS,LEU, PHE, SER, THR, TRP, or TYR; (28) the amino acid residuecorresponding to position Y37 of SEQ ID NO:6 is ARG, ASP, GLU, or PRO;(29) the amino acid residue corresponding to position E41 of SEQ ID NO:6is ALA, or GLN; (30) the amino acid residue corresponding to positionH45 of SEQ ID NO:6 is ALA, GLN, LYS, or PHE; (31) the amino acid residuecorresponding to position Q46 of SEQ ID NO:6 is ALA, ARG, SER, THR, orTRP; (32) the amino acid residue corresponding to position Q49 of SEQ IDNO:6 is HIS, ILE, PHE, or TRP; (33) the amino acid residue correspondingto position F50 of SEQ ID NO:6 is ARG, GLN, HIS, or SER; (34) the aminoacid residue corresponding to position Y53 of SEQ ID NO:6 is ALA, PHE,SER, or VAL; (35) the amino acid residue corresponding to position S54of SEQ ID NO:6 is THR; (36) the amino acid residue corresponding toposition M56 of SEQ ID NO:6 is TRP; (37) the amino acid residuecorresponding to position E59 of SEQ ID NO:6 is ARG, SER, or THR; (38)the amino acid residue corresponding to position K62 of SEQ ID NO:6 isALA, ARG, TYR, or VAL; (39) the amino acid residue corresponding toposition N63 of SEQ ID NO:6 is ARG, ASP, GLN, or GLU; (40) the aminoacid residue corresponding to position F64 of SEQ ID NO:6 is GLU, orLEU; (41) the amino acid residue corresponding to position M66 of SEQ IDNO:6 is GLU, PHE, or TYR; (42) the amino acid residue corresponding toposition K67 of SEQ ID NO:6 is ARG, ASP, GLN, GLU, SER, or THR; (43) theamino acid residue corresponding to position K70 of SEQ ID NO:6 is ARG,ASP, GLN, or LEU; (44) the amino acid residue corresponding to positionE71 of SEQ ID NO:6 is ARG, GLN, or SER; (45) the amino acid residuecorresponding to position K72 of SEQ ID NO:6 is ARG, GLN, GLU, HIS, ILE,LEU, THR, or VAL; (46) the amino acid residue corresponding to positionE75 of SEQ ID NO:6 is ARG, ASP, PRO, or SER; (47) the amino acid residuecorresponding to position E76 of SEQ ID NO:6 is ARG, LEU, or PRO; (48)the amino acid residue corresponding to position D77 of SEQ ID NO:6 isARG, ASN, or SER; (49) the amino acid residue corresponding to positionK78 of SEQ ID NO:6 is ARG, ASP, GLN, LEU, or MET; (50) the amino acidresidue corresponding to position I80 of SEQ ID NO:6 is ARG, GLN, orLEU; (51) the amino acid residue corresponding to position D84 of SEQ IDNO:6 is ALA, ASN, GLY, or PRO; (52) the amino acid residue correspondingto position H85 of SEQ ID NO:6 is ALA; (53) the amino acid residuecorresponding to position L89 of SEQ ID NO:6 is GLY, or SER; (54) theamino acid residue corresponding to position Q92 of SEQ ID NO:6 is ALA,ASP, GLY, MET, or PRO; (55) the amino acid residue corresponding toposition D93 of SEQ ID NO:6 is HIS; (56) the amino acid residuecorresponding to position E94 of SEQ ID NO:6 is ARG, GLN, LEU, or TYR;(57) the amino acid residue corresponding to position S96 of SEQ ID NO:6is ALA, ARG, or GLU; (58) the amino acid residue corresponding toposition P97 of SEQ ID NO:6 is ASP, or GLU; (59) the amino acid residuecorresponding to position T98 of SEQ ID NO:6 is ARG, GLN, GLU, ILE, TRP,or VAL; (60) the amino acid residue corresponding to position N101 ofSEQ ID NO:6 is ALA, ARG, ASP, GLN, GLU, HIS, LYS, SER, or THR; (61) theamino acid residue corresponding to position K102 of SEQ ID NO:6 is ARG,GLN, GLU, LEU, TRP, or TYR; (62) the amino acid residue corresponding toposition K104 of SEQ ID NO:6 is ARG, ASN, GLN, GLU, ILE, PHE, or VAL;(63) the amino acid residue corresponding to position D105 of SEQ IDNO:6 is GLN, GLU, or ILE; (64) the amino acid residue corresponding toposition R108 of SEQ ID NO:6 is ILE; (65) the amino acid residuecorresponding to position F110 of SEQ ID NO:6 is TYR; (66) the aminoacid residue corresponding to position S113 of SEQ ID NO:6 is ALA; (67)the amino acid residue corresponding to position Y115 of SEQ ID NO:6 isPHE; (68) the amino acid residue corresponding to position K116 of SEQID NO:6 is ARG, ASP, GLN, GLU, HIS, ILE, LEU, SER, THR, TRP, or TYR;(69) the amino acid residue corresponding to position K117 of SEQ IDNO:6 is ARG, LEU, MET, TRP, or VAL; (70) the amino acid residuecorresponding to position H119 of SEQ ID NO:6 is ALA, ASN, GLY, PRO, orSER; (71) the amino acid residue corresponding to position D121 of SEQID NO:6 is ALA, or ASN; (72) the amino acid residue corresponding toposition C122 of SEQ ID NO:6 is ALA, ARG, GLU, GLY, LYS, THR, or VAL;(73) the amino acid residue corresponding to position S123 of SEQ IDNO:6 is ALA, ARG, MET, or THR; (74) the amino acid residue correspondingto position M124 of SEQ ID NO:6 is ARG, or LYS; (75) the amino acidresidue corresponding to position P125 of SEQ ID NO:6 is ALA; (76) theamino acid residue corresponding to position L126 of SEQ ID NO:6 is CYS;(77) the amino acid residue corresponding to position S127 of SEQ IDNO:6 is ALA; (78) the amino acid residue corresponding to position D128of SEQ ID NO:6 is GLY; (79) the amino acid residue corresponding toposition D129 of SEQ ID NO:6 is ARG, GLN, GLU, LEU, or TRP; (80) theamino acid residue corresponding to position P130 of SEQ ID NO:6 is SER;(81) the amino acid residue corresponding to position K131 of SEQ IDNO:6 is ALA, ARG, ASP, GLN, GLU, LEU, SER, or TYR; (82) the amino acidresidue corresponding to position V132 of SEQ ID NO:6 is ARG, GLU, SER,THR, or TYR; (83) the amino acid residue corresponding to position P134of SEQ ID NO:6 is ALA, ASP, or HIS; (84) the amino acid residuecorresponding to position Y135 of SEQ ID NO:6 is ARG, ASN, ASP, GLN,LEU, LYS, MET, PRO, THR, or TRP; (85) the amino acid residuecorresponding to position E136 of SEQ ID NO:6 is ARG, ASP, GLN, PRO,SER, THR, or TRP; (86) the amino acid residue corresponding to positionK137 of SEQ ID NO:6 is ARG, GLN, GLU, ILE, LEU, THR, TRP, or VAL; (87)the amino acid residue corresponding to position E140 of SEQ ID NO:6 isARG, ASP, ILE, or LEU; (88) the amino acid residue corresponding toposition R141 of SEQ ID NO:6 is ASP, GLN, HIS, LEU, TRP, or TYR; (89)the amino acid residue corresponding to position T142 of SEQ ID NO:6 isGLU; (90) the amino acid residue corresponding to position R143 of SEQID NO:6 is ALA, or VAL; (91) the amino acid residue corresponding toposition E144 of SEQ ID NO:6 is ARG, ASP, GLN, or ILE; (92) the aminoacid residue corresponding to position F146 of SEQ ID NO:6 is CYS; (93)the amino acid residue corresponding to position E147 of SEQ ID NO:6 isALA, ARG, GLN, or LYS; (94) the amino acid residue corresponding toposition I148 of SEQ ID NO:6 is ASP, GLU, or VAL; (95) the amino acidresidue corresponding to position E150 of SEQ ID NO:6 is ALA, or ARG;(96) the amino acid residue corresponding to position E151 of SEQ IDNO:6 is ARG; (97) the amino acid residue corresponding to position T152of SEQ ID NO:6 is ALA, ARG, GLN, HIS, TYR, or VAL; (98) the amino acidresidue corresponding to position A153 of SEQ ID NO:6 is ARG, GLN, GLY,or SER; (99) the amino acid residue corresponding to position R154 ofSEQ ID NO:6 is GLN, GLU, PRO, SER, or THR; (100) the amino acid residuecorresponding to position K155 of SEQ ID NO:6 is ARG, ASN, ASP, GLN,GLU, GLY, HIS, PRO, THR, TRP, or TYR; (101) the amino acid residuecorresponding to position Y156 of SEQ ID NO:6 is ARG, ASP, GLN, GLU,MET, PHE, THR, or VAL; (102) the amino acid residue corresponding toposition N157 of SEQ ID NO:6 is ARG, ASP, GLU, or PRO; (103) the aminoacid residue corresponding to position F158 of SEQ ID NO:6 is ARG, ASN,GLU, LEU, THR, or VAL; (104) the amino acid residue corresponding toposition Q159 of SEQ ID NO:6 is ARG, GLU, GLY, LEU, THR, or TRP; (105)the amino acid residue corresponding to position P160 of SEQ ID NO:6 isALA, ARG, GLU, ILE, LEU, MET, PHE, SER, or THR; (106) the amino acidresidue corresponding to position V161 of SEQ ID NO:6 is GLU, ILE, TRP,or TYR; (107) the amino acid residue corresponding to position T166 ofSEQ ID NO:6 is ILE, LEU, or MET; (108) the amino acid residuecorresponding to position D167 of SEQ ID NO:6 is ALA, ASN, GLN, THR, orVAL; (109) the amino acid residue corresponding to position V168 of SEQID NO:6 is ARG, or MET; (110) the amino acid residue corresponding toposition I170 of SEQ ID NO:6 is LYS, MET, or THR; (111) the amino acidresidue corresponding to position A171 of SEQ ID NO:6 is PRO; (112) theamino acid residue corresponding to position G173 of SEQ ID NO:6 is SER;(113) the amino acid residue corresponding to position E175 of SEQ IDNO:6 is ARG, LYS, or VAL; (114) the amino acid residue corresponding toposition E176 of SEQ ID NO:6 is PHE, or TYR; (115) the amino acidresidue corresponding to position G178 of SEQ ID NO:6 is PRO; (116) theamino acid residue corresponding to position V179 of SEQ ID NO:6 is ARG,or ILE; (117) the amino acid residue corresponding to position V182 ofSEQ ID NO:6 is ALA, LYS, or PRO; (118) the amino acid residuecorresponding to position D184 of SEQ ID NO:6 is GLU; (119) the aminoacid residue corresponding to position S187 of SEQ ID NO:6 is ALA, ARG,ASN, GLN, GLU, LYS, or THR; (120) the amino acid residue correspondingto position A188 of SEQ ID NO:6 is MET, PHE, or THR; (121) the aminoacid residue corresponding to position I189 of SEQ ID NO:6 is LEU; (122)the amino acid residue corresponding to position S190 of SEQ ID NO:6 isASP, GLU, or TYR; (123) the amino acid residue corresponding to positionS191 of SEQ ID NO:6 is ALA, ARG, GLN, GLU, HIS, ILE, LEU, THR, TRP, TYR,or VAL; (124) the amino acid residue corresponding to position L192 ofSEQ ID NO:6 is HIS, or PHE; (125) the amino acid residue correspondingto position K193 of SEQ ID NO:6 is ALA, ARG, GLN, GLU, ILE, LEU, PHE, orTYR; (126) the amino acid residue corresponding to position K194 of SEQID NO:6 is ALA, ARG, ASP, GLN, GLU, LEU, SER, or THR; (127) the aminoacid residue corresponding to position Y195 of SEQ ID NO:6 is ALA, GLN,or HIS; (128) the amino acid residue corresponding to position N197 ofSEQ ID NO:6 is ALA, ARG, ASP, GLN, GLU, LEU, LYS, PRO, or SER; (129) theamino acid residue corresponding to position D198 of SEQ ID NO:6 is LYS;(130) the amino acid residue corresponding to position V199 of SEQ IDNO:6 is CYS, HIS, ILE, or LEU; (131) the amino acid residuecorresponding to position P200 of SEQ ID NO:6 is ARG, ASP, GLU, GLY,LEU, or MET; (132) the amino acid residue corresponding to position N201of SEQ ID NO:6 is ARG, GLN, GLU, or LYS; (133) the amino acid residuecorresponding to position I202 of SEQ ID NO:6 is ALA, or MET; (134) theamino acid residue corresponding to position W203 of SEQ ID NO:6 is ALA,ARG, HIS, LEU, PHE, or TYR; (135) the amino acid residue correspondingto position D204 of SEQ ID NO:6 is ARG, ASN, or SER; (136) the aminoacid residue corresponding to position R205 of SEQ ID NO:6 is GLN, orLEU; (137) the amino acid residue corresponding to position G208 of SEQID NO:6 is ALA; (138) the amino acid residue corresponding to positionV210 of SEQ ID NO:6 is PRO, or THR; (139) the amino acid residuecorresponding to position I211 of SEQ ID NO:6 is VAL; (140) the aminoacid residue corresponding to position M212 of SEQ ID NO:6 is ARG, LEU,or LYS; (141) the amino acid residue corresponding to position L213 ofSEQ ID NO:6 is MET; (142) the amino acid residue corresponding toposition I215 of SEQ ID NO:6 is VAL; (143) the amino acid residuecorresponding to position G216 of SEQ ID NO:6 is ARG, ASN, or THR; (144)the amino acid residue corresponding to position S218 of SEQ ID NO:6 isGLY, or THR; (145) the amino acid residue corresponding to position Y219of SEQ ID NO:6 is ARG, ASP, GLY, or HIS; (146) the amino acid residuecorresponding to position D220 of SEQ ID NO:6 is ARG, GLN, GLU, or THR;(147) the amino acid residue corresponding to position K221 of SEQ IDNO:6 is ARG, GLN, GLU, ILE, THR, or VAL; (148) the amino acid residuecorresponding to position V222 of SEQ ID NO:6 is ILE; (149) the aminoacid residue corresponding to position E224 of SEQ ID NO:6 is GLN, MET,or TYR; (150) the amino acid residue corresponding to position D228 ofSEQ ID NO:6 is GLU, or SER; (151) the amino acid residue correspondingto position K229 of SEQ ID NO:6 is ALA, ARG, ASN, GLN, GLU, SER, or THR;(152) the amino acid residue corresponding to position V230 of SEQ IDNO:6 is ALA; (153) the amino acid residue corresponding to position R231of SEQ ID NO:6 is ALA, GLU, LEU, or PHE; (154) the amino acid residuecorresponding to position G232 of SEQ ID NO:6 is ARG, ASP, GLU, LYS, orPRO; (155) the amino acid residue corresponding to position I233 of SEQID NO:6 is LEU; (156) the amino acid residue corresponding to positionL234 of SEQ ID NO:6 is ALA, or MET; (157) the amino acid residuecorresponding to position E235 of SEQ ID NO:6 is ARG, or TRP; (158) theamino acid residue corresponding to position V237 of SEQ ID NO:6 is LEU;(159) the amino acid residue corresponding to position K238 of SEQ IDNO:6 is ARG, GLN, GLU, SER, or TRP; (160) the amino acid residuecorresponding to position R239 of SEQ ID NO:6 is SER; (161) the aminoacid residue corresponding to position E240 of SEQ ID NO:6 is ASN, ASP,HIS, MET, THR, TYR, or VAL; (162) the amino acid residue correspondingto position D241 of SEQ ID NO:6 is ARG, ASN, GLN, GLY, or PRO; (163) theamino acid residue corresponding to position L242 of SEQ ID NO:6 is TRP;(164) the amino acid residue corresponding to position V244 of SEQ IDNO:6 is LEU; (165) the amino acid residue corresponding to position E245of SEQ ID NO:6 is ALA, or GLN; (166) the amino acid residuecorresponding to position G246 of SEQ ID NO:6 is ALA, CYS, or VAL; (167)the amino acid residue corresponding to position H247 of SEQ ID NO:6 isALA; (168) the amino acid residue corresponding to position T249 of SEQID NO:6 is SER; (169) the amino acid residue corresponding to positionD250 of SEQ ID NO:6 is ALA, or ASN; (170) the amino acid residuecorresponding to position A253 of SEQ ID NO:6 is ASN, ASP, PRO, SER, orTHR; (171) the amino acid residue corresponding to position Y255 of SEQID NO:6 is ASP, GLN, GLU, or LYS; (172) the amino acid residuecorresponding to position A256 of SEQ ID NO:6 is LEU; (173) the aminoacid residue corresponding to position R258 of SEQ ID NO:6 is GLU, orLEU; (174) the amino acid residue corresponding to position N259 of SEQID NO:6 is ALA, ARG, ASP, GLU, or TRP; (175) the amino acid residuecorresponding to position E262 of SEQ ID NO:6 is ARG, GLN, or LEU; (176)the amino acid residue corresponding to position R266 of SEQ ID NO:6 isALA, or VAL; (177) the amino acid residue corresponding to position K269of SEQ ID NO:6 is ARG, ASN, ILE, LEU, or VAL; (178) the amino acidresidue corresponding to position G271 of SEQ ID NO:6 is ALA, or ASP;(179) the amino acid residue corresponding to position A273 of SEQ IDNO:6 is TRP; (180) the amino acid residue corresponding to position R280of SEQ ID NO:6 is GLU; (181) the amino acid residue corresponding toposition G281 of SEQ ID NO:6 is ALA; (182) the amino acid residuecorresponding to position V282 of SEQ ID NO:6 is ILE; (183) the aminoacid residue corresponding to position L284 of SEQ ID NO:6 is LYS; (184)the amino acid residue corresponding to position S286 of SEQ ID NO:6 isALA, ASP, or VAL; (185) the amino acid residue corresponding to positionN287 of SEQ ID NO:6 is ARG, ASP, GLN, GLU, HIS, LEU, or PHE; (186) theamino acid residue corresponding to position D290 of SEQ ID NO:6 is ALA,ARG, ASN, GLN, GLU, LYS, or PHE; (187) the amino acid residuecorresponding to position E291 of SEQ ID NO:6 is ARG, or ASP; (188) theamino acid residue corresponding to position I292 of SEQ ID NO:6 is GLN,GLU, or LEU; (189) the amino acid residue corresponding to position E295of SEQ ID NO:6 is PRO; (190) the amino acid residue corresponding toposition R296 of SEQ ID NO:6 is ASP, GLU, or TYR; (191) the amino acidresidue corresponding to position E297 of SEQ ID NO:6 is ARG, GLN, GLY,HIS, LEU, or LYS; (192) the amino acid residue corresponding to positionS299 of SEQ ID NO:6 is ALA, or GLN; (193) the amino acid residuecorresponding to position N300 of SEQ ID NO:6 is ARG, ASP, GLN, or GLU;(194) the amino acid residue corresponding to position K302 of SEQ IDNO:6 is ARG, GLN, GLU, ILE, LEU, MET, or PRO; (195) the amino acidresidue corresponding to position R303 of SEQ ID NO:6 is ASP, GLN, orGLU; (196) the amino acid residue corresponding to position E307 of SEQID NO:6 is ARG, or GLN; (197) the amino acid residue corresponding toposition D313 of SEQ ID NO:6 is PRO; (198) the amino acid residuecorresponding to position Y315 of SEQ ID NO:6 is TRP; (199) the aminoacid residue corresponding to position K318 of SEQ ID NO:6 is ARG, ASN,ASP, or GLU; (200) the amino acid residue corresponding to position K321of SEQ ID NO:6 is ARG, ASP, GLU, HIS, SER, or THR; (201) the amino acidresidue corresponding to position D322 of SEQ ID NO:6 is THR; (202) theamino acid residue corresponding to position S323 of SEQ ID NO:6 is ASP,or GLY; (203) the amino acid residue corresponding to position K324 ofSEQ ID NO:6 is ARG, ASP, GLU, HIS, PRO, SER, or TRP; (204) the aminoacid residue corresponding to position R325 of SEQ ID NO:6 is ALA, ASN,ASP, GLN, GLU, GLY, SER, or TRP; (205) the amino acid residuecorresponding to position L326 of SEQ ID NO:6 is HIS; (206) the aminoacid residue corresponding to position E327 of SEQ ID NO:6 is ARG, ILE,or TYR; (207) the amino acid residue corresponding to position L328 ofSEQ ID NO:6 is ARG, ASP, GLU, PHE, TRP, or TYR; (208) the amino acidresidue corresponding to position I330 of SEQ ID NO:6 is LEU; (209) theamino acid residue corresponding to position W331 of SEQ ID NO:6 is GLU,HIS, LEU, or PHE; (210) the amino acid residue corresponding to positionN333 of SEQ ID NO:6 is ALA, or SER; (211) the amino acid residuecorresponding to position L334 of SEQ ID NO:6 is TYR; (212) the aminoacid residue corresponding to position R339 of SEQ ID NO:6 is ALA, GLU,GLY, or THR; (213) the amino acid residue corresponding to position Y340of SEQ ID NO:6 is ALA, ARG, HIS, PHE, or PRO; (214) the amino acidresidue corresponding to position E343 of SEQ ID NO:6 is ASN, ASP, GLN,LEU, MET, or TYR; (215) the amino acid residue corresponding to positionY344 of SEQ ID NO:6 is HIS; (216) the amino acid residue correspondingto position E345 of SEQ ID NO:6 is ASP, or PRO; (217) the amino acidresidue corresponding to position K348 of SEQ ID NO:6 is ALA, ARG, GLN,GLU, LEU, TRP, TYR, or VAL; (218) the amino acid residue correspondingto position M349 of SEQ ID NO:6 is ALA, ARG, ASN, CYS, GLN, GLU, ILE,LYS, THR, TRP, or TYR; (219) the amino acid residue corresponding toposition V350 of SEQ ID NO:6 is ALA; (220) the amino acid residuecorresponding to position N352 of SEQ ID NO:6 is ARG, GLN, GLU, LEU, orMET; (221) the amino acid residue corresponding to position K353 of SEQID NO:6 is ALA, ARG, GLN, GLU, HIS, LEU, or TRP; (222) the amino acidresidue corresponding to position E356 of SEQ ID NO:6 is ARG, or TRP;(223) the amino acid residue corresponding to position N357 of SEQ IDNO:6 is HIS; (224) the amino acid residue corresponding to position F358of SEQ ID NO:6 is LEU; (225) the amino acid residue corresponding toposition E360 of SEQ ID NO:6 is ASP; (226) the amino acid residuecorresponding to position G361 of SEQ ID NO:6 is ASP, or PRO; (227) theamino acid residue corresponding to position V362 of SEQ ID NO:6 is ILE;(228) the amino acid residue corresponding to position D363 of SEQ IDNO:6 is HIS, or PRO; (229) the amino acid residue corresponding toposition I364 of SEQ ID NO:6 is GLU, LEU, MET, PRO, or TRP; (230) theamino acid residue corresponding to position K365 of SEQ ID NO:6 is ARG,ASN, ASP, GLU, HIS, SER, or THR; (231) the amino acid residuecorresponding to position F366 of SEQ ID NO:6 is ARG, HIS, LEU, or TYR;(232) the amino acid residue corresponding to position Y368 of SEQ IDNO:6 is ARG, or LEU; (233) the amino acid residue corresponding toposition Q369 of SEQ ID NO:6 is ARG, ASP, GLU, or LEU; (234) the aminoacid residue corresponding to position Y370 of SEQ ID NO:6 is HIS; (235)the amino acid residue corresponding to position Y372 of SEQ ID NO:6 isPRO; (236) the amino acid residue corresponding to position D373 of SEQID NO:6 is ALA, ARG, ASN, GLU, or LEU; (237) the amino acid residuecorresponding to position S374 of SEQ ID NO:6 is ALA, CYS, GLN, or THR;(238) the amino acid residue corresponding to position Y375 of SEQ IDNO:6 is ALA, or TRP; (239) the amino acid residue corresponding toposition F376 of SEQ ID NO:6 is ARG, GLN, GLU, HIS, or LYS; (240) theamino acid residue corresponding to position D377 of SEQ ID NO:6 is ALA,ARG, ASN, HIS, or SER; (241) the amino acid residue corresponding toposition R379 of SEQ ID NO:6 is CYS; (242) the amino acid residuecorresponding to position E380 of SEQ ID NO:6 is ARG, ASN, ASP, or LEU;(243) the amino acid residue corresponding to position K382 of SEQ IDNO:6 is ARG, ASN, ASP, GLN, GLU, or LEU; (244) the amino acid residuecorresponding to position M383 of SEQ ID NO:6 is LEU; (245) the aminoacid residue corresponding to position K384 of SEQ ID NO:6 is ARG, ASP,GLN, GLU, LEU, SER, or THR; (246) the amino acid residue correspondingto position N385 of SEQ ID NO:6 is PRO, or THR; (247) the amino acidresidue corresponding to position D386 of SEQ ID NO:6 is HIS; (248) theamino acid residue corresponding to position R388 of SEQ ID NO:6 is GLU;(249) the amino acid residue corresponding to position E389 of SEQ IDNO:6 is ARG, or ASP; (250) the amino acid residue corresponding toposition K392 of SEQ ID NO:6 is ARG, GLU, HIS, LEU, MET, PHE, or TYR;(251) the amino acid residue corresponding to position K393 of SEQ IDNO:6 is ALA, ARG, GLN, GLU, HIS, LEU, PHE, THR, TRP, or TYR; (252) theamino acid residue corresponding to position K396 of SEQ ID NO:6 is ALA,ARG, GLN, GLU, ILE, LEU, MET, or PHE; (253) the amino acid residuecorresponding to position R397 of SEQ ID NO:6 is GLN, GLU, HIS, ILE,LEU, LYS, or SER; (254) the amino acid residue corresponding to positionE400 of SEQ ID NO:6 is ARG, GLN, LEU, or TYR; (255) the amino acidresidue corresponding to position S403 of SEQ ID NO:6 is ALA, ARG, GLU,HIS, LEU, PHE, THR, TRP, or TYR; (256) the amino acid residuecorresponding to position N407 of SEQ ID NO:6 is ARG, ASP, GLN, GLU,HIS, ILE, LEU, PHE, TRP, or TYR; or (257) the amino acid residuecorresponding to position L408 of SEQ ID NO:6 is ARG, ASN, ASP, GLY,THR, or TRP; or combinations thereof. 4.-244. (canceled)
 245. Thepolypeptide of claim 1, wherein the polypeptide is capable of convertingfructose to tagatose through epimerization at the carbon-4 position offructose.
 246. The polypeptide of claim 1, wherein the polypeptide is arecombinant polypeptide.
 247. The polypeptide of claim 1, wherein thepolypeptide is immobilized to a carrier or support.
 248. (canceled) 249.The polypeptide of claim 247, wherein the carrier or support is anorganic composition.
 250. (canceled)
 251. The polypeptide of claim 249,wherein the organic composition is functionalized with a chemical group.252. (canceled)
 253. The polypeptide of claim 247, wherein the carrieror support is an inorganic composition. 254.-256. (canceled)
 257. Amodified microorganism expressing the polypeptide of claim
 1. 258.-259.(canceled)
 260. A method of producing tagatose, the method comprising:(a) contacting fructose with the polypeptide of claim 1 or amicroorganism expressing the polypeptide; and (b) converting fructose totagatose.
 261. (canceled)
 262. A method for producing a tagatosecomposition, comprising the steps of: (a) providing a startingcomposition comprising greater than about 0.3%, about 0.5%, about 1%,about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90% offructose by weight; (b) contacting the starting composition with thepolypeptide of claim 1 or a microorganism expressing the polypeptide;and (c) producing a tagatose composition comprising tagatose.
 263. Themethod of claim 262, wherein the tagatose composition comprises tagatosein an amount greater than about 0.1%, 0.2%, 0.3%, about 0.5%, about 1%,about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, or about 40%.
 264. The method of claim 262, wherein the tagatosecomposition comprises tagatose in an amount between about 0.3-80 wt %.265. (canceled)
 266. The method of claim 262, wherein the startingcomposition comprises fructose in an amount between about 0.30% to about70% by weight. 267.-269. (canceled)
 270. The method of claim 260,wherein the polypeptide or the microorganism expressing the polypeptideis immobilized to a carrier or support.
 271. (canceled)
 272. The methodof claim 270, wherein the carrier or support is an organic composition.273. (canceled)
 274. The method of claim 272, wherein the organiccomposition is functionalized with a chemical group.
 275. (canceled)276. The method of claim 270, wherein the carrier or support is aninorganic composition. 277.-279. (canceled)
 280. The method of claim260, wherein contacting fructose with the polypeptide or themicroorganism expressing the polypeptide is performed at a temperaturebetween about 40° C. and about 100° C.
 281. (canceled)
 282. The methodof claim 260, wherein the contacting of fructose with the polypeptide orthe microorganism expressing the polypeptide is performed at betweenabout pH 4.5 and about pH
 8. 283. (canceled)
 284. The method of claim260, wherein the contacting of fructose with the polypeptide or themicroorganism expressing the polypeptide is performed in the presence ofa metal ion.
 285. The method of claim 284, wherein the metal ion is adivalent metal cation. 286.-289. (canceled)
 290. The method of claim260, wherein the tagatose is isolated from the feed using achromatographic method.
 291. The method of claim 290, wherein thechromatic method is simulated moving bed chromatography.
 292. The methodof claim 260, wherein the tagatose product is crystallized.
 293. Themethod of claim 292, wherein the crystallization is isothermalevaporative crystallization or evaporative cooling crystallization. 294.The method of claim 260, wherein the fructose is produced from glucoseor sucrose.